Isolated human synthase proteins

ABSTRACT

The present invention provides amino acid sequences of peptides that are encoded by genes within the human genome, the enzyme peptides of the present invention. The present invention specifically provides isolated peptide and nucleic acid molecules, methods of identifying orthologs and paralogs of the enzyme peptides, and methods of identifying modulators of the enzyme peptides.

RELATED APPLICATIONS

The present application is a divisional of U.S. application Ser. No.09/819,993, filed on Mar. 29, 2001 and issued on Aug. 20, 2002 as U.S.Pat. No. 6,436,692.

FIELD OF THE INVENTION

The present invention is in the field of enzyme proteins that arerelated to the synthase enzyme subfamily, recombinant DNA molecules, andprotein production. The present invention specifically provides novelpeptides and proteins and nucleic acid molecules encoding such peptideand protein molecules, all of which are useful in the development ofhuman therapeutics and diagnostic compositions and methods.

BACKGROUND OF THE INVENTION

Many human enzymes serve as targets for the action of pharmaceuticallyactive compounds. Several classes of human enzymes that serve as suchtargets include helicase, steroid esterase and sulfatase, convertase,synthase, dehydrogenase, monoxygenase, transferase, kinase, glutanase,decarboxylase, isomerase and reductase. It is therefore important indeveloping new pharmaceutical compounds to identify target enzymeproteins that can be put into high-throughput screening formats. Thepresent invention advances the state of the art by providing novel humandrug target enzymes related to the synthase subfamily.

Synthases

The novel human protein, and encoding gene, provided by the presentinvention is related to the family of synthase enzymes in general, andshows the greatest degree of similarity to human cytoplasmic3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase. Furthermore,the protein of the present invention may be an alternative splice formof the HMG-CoA synthase enzyme provided in Genbank gi4504429 (see theamino acid sequence alignment in FIG. 2). HMG-CoA synthase, along withHMG-CoA reductase which is also found on human chromosome 5, is atranscriptionally regulated enzyme that is important incholesterologenesis.

Mutation of Cys129 to serine or alanine has been shown to abolishHMG-CoA synthase activity by interrupting the first catalytic step,enzyme acetylation by acetyl coenzyme A, in HMG-COA synthesis (Rokosz etal., Arch. Biochem. Biophys. 312 (1), 1-13 (1994)). A beta-lactoneinhibitor compound known as L-659,699, is a strong inhibitor of HMG-CoAsynthase (Rokosz et al., Arch. Biochem. Biophys. 312 (1), 1-13 (1994)).

For a further review of HMG-CoA synthase, see Mehrabian et al., J. BiolChem 1986 December 5;261(34):16249-55; Ayte et al., Proc. Nat. Acad.Sci. 87: 3874-3878, 1990; Gil et al., Proc. Nat. Acad. Sci. 84:1863-1866, 1987; Leonard et al., Proc. Nat. Acad. Sci. 83: 2187-2189,1986; and Russ et al., Biochim. Biophys. Acta 1132: 329-331, 1992.

Due to their importance in cholesterologenesis, novel human HMG-CoAsynthase proteins/genes, such as provided by the present invention, arevaluable as potential targets for the development of therapeutics totreat cholesterol-related diseases/disorders. Furthermore, SNPs inHMG-CoA synthase genes, such as provided by the present invention, arevaluable markers for the diagnosis, prognosis, prevention, and/ortreatment of cholesterol-related diseases/disorders.

Using the information provided by the present invention, reagents suchas probes/primers for detecting the SNPs or the expression of theprotein/gene provided herein may be readily developed and, if desired,incorporated into kit formats such as nucleic acid arrays, primerextension reactions coupled with mass spec detection (for SNPdetection), or TaqMan PCR assays (Applied Biosystems, Foster City,Calif.).

Enzyme proteins, particularly members of the synthase enzyme subfamily,are a major target for drug action and development. Accordingly, it isvaluable to the field of pharmaceutical development to identify andcharacterize previously unknown members of this subfamily of enzymeproteins. The present invention advances the state of the art byproviding previously unidentified human enzyme proteins, and thepolynucleotides encoding them, that have homology to members of thesynthase enzyme subfamily. These novel compositions are useful in thediagnosis, prevention and treatment of biological processes associatedwith human diseases.

SUMMARY OF THE INVENTION

The present invention is based in part on the identification of aminoacid sequences of human enzyme peptides and proteins that are related tothe synthase enzyme subfamily, as well as allelic variants and othermammalian orthologs thereof. These unique peptide sequences, and nucleicacid sequences that encode these peptides, can be used as models for thedevelopment of human therapeutic targets, aid in the identification oftherapeutic proteins, and serve as targets for the development of humantherapeutic agents that modulate enzyme activity in cells and tissuesthat express the enzyme. Experimental data as provided in FIG. 1indicates expression in humans in teratocarcinoma and teratocarcinomaneuronal precursor cells, fetal brain, liver and liver adenocarcinoma,lung small cell carinoma, and the genitourinary tract.

DESCRIPTION OF THE FIGURE SHEETS

FIGS. 1A-1C provides the nucleotide sequence of a cDNA molecule thatencodes the enzyme protein of the present invention. (SEQ ID NO:1) Inaddition, structure and functional information is provided, such as ATGstart, stop and tissue distribution, where available, that allows one toreadily determine specific uses of inventions based on this molecularsequence. Experimental data as provided in FIG. 1 indicates expressionin humans in teratocarcinoma and teratocarcinoma neuronal precursorcells, fetal brain, liver and liver adenocarcinoma, lung small cellcarinoma, and the genitourinary tract.

FIGS. 2A-2E provides the predicted amino acid sequence of the enzyme ofthe present invention. (SEQ ID NO:2) In addition structure andfunctional information such as protein family, function, andmodification sites is provided where available, allowing one to readilydetermine specific uses of inventions based on this molecular sequence.

FIGS. 3A-3R provides genomic sequences that span the gene encoding theenzyme protein of the present invention. (SEQ ID NO:3) In additionstructure and functional information, such as intron/exon structure,promoter location, etc., is provided where available, allowing one toreadily determine specific uses of inventions based on this molecularsequence. As illustrated in FIG. 3, SNPs were identified at 16 differentnucleotide positions.

DETAILED DESCRIPTION OF THE INVENTION General Description

The present invention is based on the sequencing of the human genome.During the sequencing and assembly of the human genome, analysis of thesequence information revealed previously unidentified fragments of thehuman genome that encode peptides that share structural and/or sequencehomology to protein/peptide/domains identified and characterized withinthe art as being a enzyme protein or part of a enzyme protein and arerelated to the synthase enzyme subfamily. Utilizing these sequences,additional genomic sequences were assembled and transcript and/or cDNAsequences were isolated and characterized. Based on this analysis, thepresent invention provides amino acid sequences of human enzyme peptidesand proteins that are related to the synthase enzyme subfamily, nucleicacid sequences in the form of transcript sequences, cDNA sequencesand/or genomic sequences that encode these enzyme peptides and proteins,nucleic acid variation (allelic information), tissue distribution ofexpression, and information about the closest art knownprotein/peptide/domain that has structural or sequence homology to theenzyme of the present invention.

In addition to being previously unknown, the peptides that are providedin the present invention are selected based on their ability to be usedfor the development of commercially important products and services.Specifically, the present peptides are selected based on homology and/orstructural relatedness to known enzyme proteins of the synthase enzymesubfamily and the expression pattern observed. Experimental data asprovided in FIG. 1 indicates expression in humans in teratocarcinoma andteratocarcinoma neuronal precursor cells, fetal brain, liver and liveradenocarcinoma, lung small cell carinoma, and the genitourinary tract.The art has clearly established the commercial importance of members ofthis family of proteins and proteins that have expression patternssimilar to that of the present gene. Some of the more specific featuresof the peptides of the present invention, and the uses thereof, aredescribed herein, particularly in the Background of the Invention and inthe annotation provided in the Figures, and/or are known within the artfor each of the known synthase family or subfamily of enzyme proteins.

Specific Embodiments Peptide Molecules

The present invention provides nucleic acid sequences that encodeprotein molecules that have been identified as being members of theenzyme family of proteins and are related to the synthase enzymesubfamily (protein sequences are provided in FIG. 2, transcript/cDNAsequences are provided in FIG. 1 and genomic sequences are provided inFIG. 3). The peptide sequences provided in FIG. 2, as well as theobvious variants described herein, particularly allelic variants asidentified herein and using the information in FIG. 3, will be referredherein as the enzyme peptides of the present invention, enzyme peptides,or peptides/proteins of the present invention.

The present invention provides isolated peptide and protein moleculesthat consist of, consist essentially of, or comprise the amino acidsequences of the enzyme peptides disclosed in the FIG. 2, (encoded bythe nucleic acid molecule shown in FIG. 1, transcript/cDNA or FIG. 3,genomic sequence), as well as all obvious variants of these peptidesthat are within the art to make and use. Some of these variants aredescribed in detail below.

As used herein, a peptide is said to be “isolated” or “purified” when itis substantially free of cellular material or free of chemicalprecursors or other chemicals. The peptides of the present invention canbe purified to homogeneity or other degrees of purity. The level ofpurification will be based on the intended use. The critical feature isthat the preparation allows for the desired function of the peptide,even if in the presence of considerable amounts of other components (thefeatures of an isolated nucleic acid molecule is discussed below).

In some uses, “substantially free of cellular material” includespreparations of the peptide having less than about 30% (by dry weight)other proteins (i.e., contaminating protein), less than about 20% otherproteins, less than about 10% other proteins, or less than about 5%other proteins. When the peptide is recombinantly produced, it can alsobe substantially free of culture medium, i.e., culture medium representsless than about 20% of the volume of the protein preparation.

The language “substantially free of chemical precursors or otherchemicals” includes preparations of the peptide in which it is separatedfrom chemical precursors or other chemicals that are involved in itssynthesis. In one embodiment, the language “substantially free ofchemical precursors or other chemicals” includes preparations of theenzyme peptide having less than about 30% (by dry weight) chemicalprecursors or other chemicals, less than about 20% chemical precursorsor other chemicals, less than about 10% chemical precursors or otherchemicals, or less than about 5% chemical precursors or other chemicals.

The isolated enzyme peptide can be purified from cells that naturallyexpress it, purified from cells that have been altered to express it(recombinant), or synthesized using known protein synthesis methods.Experimental data as provided in FIG. 1 indicates expression in humansin teratocarcinoma and teratocarcinoma neuronal precursor cells, fetalbrain, liver and liver adenocarcinoma, lung small cell carinoma, and thegenitourinary tract. For example, a nucleic acid molecule encoding theenzyme peptide is cloned into an expression vector, the expressionvector introduced into a host cell and the protein expressed in the hostcell. The protein can then be isolated from the cells by an appropriatepurification scheme using standard protein purification techniques. Manyof these techniques are described in detail below.

Accordingly, the present invention provides proteins that consist of theamino acid sequences provided in FIG. 2 (SEQ ID NO:2), for example,proteins encoded by the transcript/cDNA nucleic acid sequences shown inFIG. 1 (SEQ ID NO:1) and the genomic sequences provided in FIG. 3 (SEQID NO:3). The amino acid sequence of such a protein is provided in FIG.2. A protein consists of an amino acid sequence when the amino acidsequence is the final amino acid sequence of the protein.

The present invention further provides proteins that consist essentiallyof the amino acid sequences provided in FIG. 2 (SEQ ID NO:2), forexample, proteins encoded by the transcript/cDNA nucleic acid sequencesshown in FIG. 1 (SEQ ID NO:1) and the genomic sequences provided in FIG.3 (SEQ ID NO:3). A protein consists essentially of an amino acidsequence when such an amino acid sequence is present with only a fewadditional amino acid residues, for example from about 1 to about 100 orso additional residues, typically from 1 to about 20 additional residuesin the final protein.

The present invention further provides proteins that comprise the aminoacid sequences provided in FIG. 2 (SEQ ID NO:2), for example, proteinsencoded by the transcript/cDNA nucleic acid sequences shown in FIG. 1(SEQ ID NO:1) and the genomic sequences provided in FIG. 3 (SEQ IDNO:3). A protein comprises an amino acid sequence when the amino acidsequence is at least part of the final amino acid sequence of theprotein. In such a fashion, the protein can be only the peptide or haveadditional amino acid molecules, such as amino acid residues (contiguousencoded sequence) that are naturally associated with it or heterologousamino acid residues/peptide sequences. Such a protein can have a fewadditional amino acid residues or can comprise several hundred or moreadditional amino acids. The preferred classes of proteins that arecomprised of the enzyme peptides of the present invention are thenaturally occurring mature proteins. A brief description of how varioustypes of these proteins can be made/isolated is provided below.

The enzyme peptides of the present invention can be attached toheterologous sequences to form chimeric or fusion proteins. Suchchimeric and fusion proteins comprise a enzyme peptide operativelylinked to a heterologous protein having an amino acid sequence notsubstantially homologous to the enzyme peptide. “Operatively linked”indicates that the enzyme peptide and the heterologous protein are fusedin-frame. The heterologous protein can be fused to the N-terminus orC-terminus of the enzyme peptide.

In some uses, the fusion protein does not affect the activity of theenzyme peptide per se. For example, the fusion protein can include, butis not limited to, enzymatic fusion proteins, for examplebeta-galactosidase fusions, yeast two-hybrid GAL fusions, poly-Hisfusions, MYC-tagged, HI-tagged and Ig fusions. Such fusion proteins,particularly poly-His fusions, can facilitate the purification ofrecombinant enzyme peptide. In certain host cells (e.g., mammalian hostcells), expression and/or secretion of a protein can be increased byusing a heterologous signal sequence.

A chimeric or fusion protein can be produced by standard recombinant DNAtechniques. For example, DNA fragments coding for the different proteinsequences are ligated together in-frame in accordance with conventionaltechniques. In another embodiment, the fusion gene can be synthesized byconventional techniques including automated DNA synthesizers.Alternatively, PCR amplification of gene fragments can be carried outusing anchor primers which give rise to complementary overhangs betweentwo consecutive gene fragments which can subsequently be annealed andre-amplified to generate a chimeric gene sequence (see Ausubel et al.,Current Protocols in Molecular Biology, 1992). Moreover, many expressionvectors are commercially available that already encode a fusion moiety(e.g., a GST protein). A enzyme peptide-encoding nucleic acid can becloned into such an expression vector such that the fusion moiety islinked in-frame to the enzyme peptide.

As mentioned above, the present invention also provides and enablesobvious variants of the amino acid sequence of the proteins of thepresent invention, such as naturally occurring mature forms of thepeptide, allelic/sequence variants of the peptides, non-naturallyoccurring recombinantly derived variants of the peptides, and orthologsand paralogs of the peptides. Such variants can readily be generatedusing art-known techniques in the fields of recombinant nucleic acidtechnology and protein biochemistry. It is understood, however, thatvariants exclude any amino acid sequences disclosed prior to theinvention.

Such variants can readily be identified/made using molecular techniquesand the sequence information disclosed herein. Further, such variantscan readily be distinguished from other peptides based on sequenceand/or structural homology to the enzyme peptides of the presentinvention. The degree of homology/identity present will be basedprimarily on whether the peptide is a functional variant ornon-functional variant, the amount of divergence present in the paralogfamily and the evolutionary distance between the orthologs.

To determine the percent identity of two amino acid sequences or twonucleic acid sequences, the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in one or both of a first and asecond amino acid or nucleic acid sequence for optimal alignment andnon-homologous sequences can be disregarded for comparison purposes). Ina preferred embodiment, at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% ormore of the length of a reference sequence is aligned for comparisonpurposes. The amino acid residues or nucleotides at corresponding aminoacid positions or nucleotide positions are then compared. When aposition in the first sequence is occupied by the same amino acidresidue or nucleotide as the corresponding position in the secondsequence, then the molecules are identical at that position (as usedherein amino acid or nucleic acid “identity” is equivalent to amino acidor nucleic acid “homology”). The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences, taking into account the number of gaps, and the length ofeach gap, which need to be introduced for optimal alignment of the twosequences.

The comparison of sequences and determination of percent identity andsimilarity between two sequences can be accomplished using amathematical algorithm. (Computational Molecular Biology, Lesk, A. M.,ed., Oxford University Press, New York, 1988; Biocomputing: Informaticsand Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993;Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and Griffin,H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis inMolecular Biology, von Heinje, G., Academic Press, 1987; and SequenceAnalysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press,New York, 1991). In a preferred embodiment, the percent identity betweentwo amino acid sequences is determined using the Needleman and Wunsch(J. Mol. Biol. (48):444-453 (1970)) algorithm which has beenincorporated into the GAP program in the GCG software package (availableat gcg.com), using either a Blossom 62 matrix or a PAM250 matrix, and agap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3,4, 5, or 6. In yet another preferred embodiment, the percent identitybetween two nucleotide sequences is determined using the GAP program inthe GCG software package (Devereux, J., et al., Nucleic Acids Res.12(1):387 (1984)) (available at .gcg.com), using a NWSgapdna.CMP matrixand a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2,3, 4, 5, or 6. In another embodiment, the percent identity between twoamino acid or nucleotide sequences is determined using the algorithm ofE. Myers and W. Miller (CABIOS, 4:11-17 (1989)) which has beenincorporated into the ALIGN program (version 2.0), using a PAM120 weightresidue table, a gap length penalty of 12 and a gap penalty of 4.

The nucleic acid and protein sequences of the present invention canfurther be used as a “query sequence” to perform a search againstsequence databases to, for example, identify other family members orrelated sequences. Such searches can be performed using the NBLAST andXBLAST programs (version 2.0) of Altschul, et al. (J. Mol. Biol.215:403-10 (1990)). BLAST nucleotide searches can be performed with theNBLAST program, score=100, wordlength=12 to obtain nucleotide sequenceshomologous to the nucleic acid molecules of the invention. BLAST proteinsearches can be performed with the XBLAST program, score=50,wordlength=3 to obtain amino acid sequences homologous to the proteinsof the invention. To obtain gapped alignments for comparison purposes,Gapped BLAST can be utilized as described in Altschul et al. (NucleicAcids Res. 25(17):3389-3402 (1997)). When utilizing BLAST and gappedBLAST programs, the default parameters of the respective programs (e.g.,XBLAST and NBLAST) can be used.

Full-length pre-processed forms, as well as mature processed forms, ofproteins that comprise one of the peptides of the present invention canreadily be identified as having complete sequence identity to one of theenzyme peptides of the present invention as well as being encoded by thesame genetic locus as the enzyme peptide provided herein. The geneencoding the novel enzyme of the present invention is located on agenome component that has been mapped to human chromosome 5 (asindicated in FIG. 3), which is supported by multiple lines of evidence,such as STS and BAC map data.

Allelic variants of a enzyme peptide can readily be identified as beinga human protein having a high degree (significant) of sequencehomology/identity to at least a portion of the enzyme peptide as well asbeing encoded by the same genetic locus as the enzyme peptide providedherein. Genetic locus can readily be determined based on the genomicinformation provided in FIG. 3, such as the genomic sequence mapped tothe reference human. The gene encoding the novel enzyme of the presentinvention is located on a genome component that has been mapped to humanchromosome 5 (as indicated in FIG. 3), which is supported by multiplelines of evidence, such as STS and BAC map data. As used herein, twoproteins (or a region of the proteins) have significant homology whenthe amino acid sequences are typically at least about 70-80%, 80-90%,and more typically at least about 90-95% or more homologous. Asignificantly homologous amino acid sequence, according to the presentinvention, will be encoded by a nucleic acid sequence that willhybridize to a enzyme peptide encoding nucleic acid molecule understringent conditions as more fully described below.

FIG. 3 provides information on SNPs that have been found in the geneencoding the enzyme of the present invention. SNPs were identified at 16different nucleotide positions. Some of these SNPs that are locatedoutside the ORF and in introns may affect gene transcription.

Paralogs of a enzyme peptide can readily be identified as having somedegree of significant sequence homology/identity to at least a portionof the enzyme peptide, as being encoded by a gene from humans, and ashaving similar activity or function. Two proteins will typically beconsidered paralogs when the amino acid sequences are typically at leastabout 60% or greater, and more typically at least about 70% or greaterhomology through a given region or domain. Such paralogs will be encodedby a nucleic acid sequence that will hybridize to a enzyme peptideencoding nucleic acid molecule under moderate to stringent conditions asmore fully described below.

Orthologs of a enzyme peptide can readily be identified as having somedegree of significant sequence homology/identity to at least a portionof the enzyme peptide as well as being encoded by a gene from anotherorganism. Preferred orthologs will be isolated from mammals, preferablyprimates, for the development of human therapeutic targets and agents.Such orthologs will be encoded by a nucleic acid sequence that willhybridize to a enzyme peptide encoding nucleic acid molecule undermoderate to stringent conditions, as more fully described below,depending on the degree of relatedness of the two organisms yielding theproteins.

Non-naturally occurring variants of the enzyme peptides of the presentinvention can readily be generated using recombinant techniques. Suchvariants include, but are not limited to deletions, additions andsubstitutions in the amino acid sequence of the enzyme peptide. Forexample, one class of substitutions are conserved amino acidsubstitution. Such substitutions are those that substitute a given aminoacid in a enzyme peptide by another amino acid of like characteristics.Typically seen as conservative substitutions are the replacements, onefor another, among the aliphatic amino acids Ala, Val, Leu, and Ile;interchange of the hydroxyl residues Ser and Thr; exchange of the acidicresidues Asp and Glu; substitution between the amide residues Asn andGln; exchange of the basic residues Lys and Arg; and replacements amongthe aromatic residues Phe and Tyr. Guidance concerning which amino acidchanges are likely to be phenotypically silent are found in Bowie etal., Science 247:1306-1310 (1990).

Variant enzyme peptides can be fully functional or can lack function inone or more activities, e.g. ability to bind substrate, ability tophosphorylate substrate, ability to mediate signaling, etc. Fullyfunctional variants typically contain only conservative variation orvariation in non-critical residues or in non-critical regions. FIG. 2provides the result of protein analysis and can be used to identifycritical domains/regions. Functional variants can also containsubstitution of similar amino acids that result in no change or aninsignificant change in function. Alternatively, such substitutions maypositively or negatively affect function to some degree.

Non-functional variants typically contain one or more non-conservativeamino acid substitutions, deletions, insertions, inversions, ortruncation or a substitution, insertion, inversion, or deletion in acritical residue or critical region.

Amino acids that are essential for function can be identified by methodsknown in the art, such as site-directed mutagenesis or alanine-scanningmutagenesis (Cunningham et al., Science 244:1081-1085 (1989)),particularly using the results provided in FIG. 2. The latter procedureintroduces single alanine mutations at every residue in the molecule.The resulting mutant molecules are then tested for biological activitysuch as enzyme activity or in assays such as an in vitro proliferativeactivity. Sites that are critical for binding partner/substrate bindingcan also be determined by structural analysis such as crystallization,nuclear magnetic resonance or photoaffinity labeling (Smith et al., .JMol. Biol. 224:899-904 (1992); de Vos et al. Science 255:306-312(1992)).

The present invention further provides fragments of the enzyme peptides,in addition to proteins and peptides that comprise and consist of suchfragments, particularly those comprising the residues identified in FIG.2. The fragments to which the invention pertains, however, are not to beconstrued as encompassing fragments that may be disclosed publicly priorto the present invention.

As used herein, a fragment comprises at least 8, 10, 12, 14, 16, or morecontiguous amino acid residues from a enzyme peptide. Such fragments canbe chosen based on the ability to retain one or more of the biologicalactivities of the enzyme peptide or could be chosen for the ability toperform a function, e.g. bind a substrate or act as an immunogen.Particularly important fragments are biologically active fragments,peptides that are, for example, about 8 or more amino acids in length.Such fragments will typically comprise a domain or motif of the enzymepeptide, e.g., active site, a transmembrane domain or asubstrate-binding domain. Further, possible fragments include, but arenot limited to, domain or motif containing fragments, soluble peptidefragments, and fragments containing immunogenic structures. Predicteddomains and functional sites are readily identifiable by computerprograms well known and readily available to those of skill in the art(e.g., PROSITE analysis). The results of one such analysis are providedin FIG. 2.

Polypeptides often contain amino acids other than the 20 amino acidscommonly referred to as the 20 naturally occurring amino acids. Further,many amino acids, including the terminal amino acids, may be modified bynatural processes, such as processing and other post-translationalmodifications, or by chemical modification techniques well known in theart. Common modifications that occur naturally in enzyme peptides aredescribed in basic texts, detailed monographs, and the researchliterature, and they are well known to those of skill in the art (someof these features are identified in FIG. 2).

Known modifications include, but are not limited to, acetylation,acylation, ADP-ribosylation, amidation, covalent attachment of flavin,covalent attachment of a heme moiety, covalent attachment of anucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of phosphotidylinositol,cross-linking, cyclization, disulfide bond formation, demethylation,formation of covalent crosslinks, formation of cystine, formation ofpyroglutamate, formylation, gamma carboxylation, glycosylation, GPIanchor formation, hydroxylation, iodination, methylation,myristoylation, oxidation, proteolytic processing, phosphorylation,prenylation, racemization, selenoylation, sulfation, transfer-RNAmediated addition of amino acids to proteins such as arginylation, andubiquitination.

Such modifications are well known to those of skill in the art and havebeen described in great detail in the scientific literature. Severalparticularly common modifications, glycosylation, lipid attachment,sulfation, gamma-carboxylation of glutamic acid residues, hydroxylationand ADP-ribosylation, for instance, are described in most basic texts,such as Proteins—Structure and Molecular Properties, 2nd Ed., T. E.Creighton, W.H. Freeman and Company, New York (1993). Many detailedreviews are available on this subject, such as by Wold, F.,Posttranslational Covalent Modification of Proteins, B. C. Johnson, Ed.,Academic Press, New York 1-12 (1983); Seifter et al. (Meth. Enzymol.182: 626-646 (1990)) and Rattan et al. (Ann. N.Y. Acad. Sci. 663:48-62(1992)).

Accordingly, the enzyme peptides of the present invention also encompassderivatives or analogs in which a substituted amino acid residue is notone encoded by the genetic code, in which a substituent group isincluded, in which the mature enzyme peptide is fused with anothercompound, such as a compound to increase the half-life of the enzymepeptide (for example, polyethylene glycol), or in which the additionalamino acids are fused to the mature enzyme peptide, such as a leader orsecretory sequence or a sequence for purification of the mature enzymepeptide or a pro-protein sequence.

Protein/Peptide Uses

The proteins of the present invention can be used in substantial andspecific assays related to the functional information provided in theFigures; to raise antibodies or to elicit another immune response; as areagent (including the labeled reagent) in assays designed toquantitatively determine levels of the protein (or its binding partneror ligand) in biological fluids; and as markers for tissues in which thecorresponding protein is preferentially expressed (either constitutivelyor at a particular stage of tissue differentiation or development or ina disease state). Where the protein binds or potentially binds toanother protein or ligand (such as, for example, in a enzyme-effectorprotein interaction or enzyme-ligand interaction), the protein can beused to identify the binding partner/ligand so as to develop a system toidentify inhibitors of the binding interaction. Any or all of these usesare capable of being developed into reagent grade or kit format forcommercialization as commercial products.

Methods for performing the uses listed above are well known to thoseskilled in the art. References disclosing such methods include“Molecular Cloning: A Laboratory Manual”, 2d ed., Cold Spring HarborLaboratory Press, Sambrook, J., E. F. Fritsch and T. Maniatis eds.,1989, and “Methods in Enzymology: Guide to Molecular CloningTechniques”, Academic Press, Berger, S. L. and A. R. Kimmel eds., 1987.

The potential uses of the peptides of the present invention are basedprimarily on the source of the protein as well as the class/action ofthe protein. For example, enzymes isolated from humans and theirhuman/mammalian orthologs serve as targets for identifying agents foruse in mammalian therapeutic applications, e.g. a human drug,particularly in modulating a biological or pathological response in acell or tissue that expresses the enzyme. Experimental data as providedin FIG. 1 indicates that the enzymes of the present invention areexpressed in humans in teratocarcinoma and teratocarcinoma neuronalprecursor cells, fetal brain, liver adenocarcinoma, lung small cellcarinoma, and the genitourinary tract, as indicated by virtual northernblot analysis. In addition, PCR-based tissue screening panels indicateexpression in liver. A large percentage of pharmaceutical agents arebeing developed that modulate the activity of enzyme proteins,particularly members of the synthase subfamily (see Background of theInvention). The structural and functional information provided in theBackground and Figures provide specific and substantial uses for themolecules of the present invention, particularly in combination with theexpression information provided in FIG. 1. Experimental data as providedin FIG. 1 indicates expression in humans in teratocarcinoma andteratocarcinoma neuronal precursor cells, fetal brain, liver and liveradenocarcinoma, lung small cell carinoma, and the genitourinary tract.Such uses can readily be determined using the information providedherein, that which is known in the art, and routine experimentation.

The proteins of the present invention (including variants and fragmentsthat may have been disclosed prior to the present invention) are usefulfor biological assays related to enzymes that are related to members ofthe synthase subfamily. Such assays involve any of the known enzymefunctions or activities or properties useful for diagnosis and treatmentof enzyme-related conditions that are specific for the subfamily ofenzymes that the one of the present invention belongs to, particularlyin cells and tissues that express the enzyme. Experimental data asprovided in FIG. 1 indicates that the enzymes of the present inventionare expressed in humans in teratocarcinoma and teratocarcinoma neuronalprecursor cells, fetal brain, liver adenocarcinoma, lung small cellcarinoma, and the genitourinary tract, as indicated by virtual northernblot analysis. In addition, PCR-based tissue screening panels indicateexpression in liver.

The proteins of the present invention are also useful in drug screeningassays, in cell-based or cell-free systems. Cell-based systems can benative, i.e., cells that normally express the enzyme, as a biopsy orexpanded in cell culture. Experimental data as provided in FIG. 1indicates expression in humans in teratocarcinoma and teratocarcinomaneuronal precursor cells, fetal brain, liver and liver adenocarcinoma,lung small cell carinoma, and the genitourinary tract. In an alternateembodiment, cell-based assays involve recombinant host cells expressingthe enzyme protein.

The polypeptides can be used to identify compounds that modulate enzymeactivity of the protein in its natural state or an altered form thatcauses a specific disease or pathology associated with the enzyme. Boththe enzymes of the present invention and appropriate variants andfragments can be used in high-throughput screens to assay candidatecompounds for the ability to bind to the enzyme. These compounds can befurther screened against a functional enzyme to determine the effect ofthe compound on the enzyme activity. Further, these compounds can betested in animal or invertebrate systems to determineactivity/effectiveness. Compounds can be identified that activate(agonist) or inactivate (antagonist) the enzyme to a desired degree.

Further, the proteins of the present invention can be used to screen acompound for the ability to stimulate or inhibit interaction between theenzyme protein and a molecule that normally interacts with the enzymeprotein, e.g. a substrate or a component of the signal pathway that theenzyme protein normally interacts (for example, another enzyme). Suchassays typically include the steps of combining the enzyme protein witha candidate compound under conditions that allow the enzyme protein, orfragment, to interact with the target molecule, and to detect theformation of a complex between the protein and the target or to detectthe biochemical consequence of the interaction with the enzyme proteinand the target, such as any of the associated effects of signaltransduction such as protein phosphorylation, cAMP turnover, andadenylate cyclase activation, etc.

Candidate compounds include, for example, 1) peptides such as solublepeptides, including Ig-tailed fusion peptides and members of randompeptide libraries (see, e.g., Lam et al., Nature 354:82-84 (1991);Houghten et al., Nature 354:84-86 (1991)) and combinatorialchemistry-derived molecular libraries made of D- and/or L-configurationamino acids; 2) phosphopeptides (e.g., members of random and partiallydegenerate, directed phosphopeptide libraries, see, e.g., Songyang etal., Cell 72:767-778 (1993)); 3) antibodies (e.g., polyclonal,monoclonal, humanized, anti-idiotypic, chimeric, and single chainantibodies as well as Fab, F(ab′)₂, Fab expression library fragments,and epitope-binding fragments of antibodies); and 4) small organic andinorganic molecules (e.g., molecules obtained from combinatorial andnatural product libraries).

One candidate compound is a soluble fragment of the receptor thatcompetes for substrate binding. Other candidate compounds include mutantenzymes or appropriate fragments containing mutations that affect enzymefunction and thus compete for substrate. Accordingly, a fragment thatcompetes for substrate, for example with a higher affinity, or afragment that binds substrate but does not allow release, is encompassedby the invention.

The invention further includes other end point assays to identifycompounds that modulate (stimulate or inhibit) enzyme activity. Theassays typically involve an assay of events in the signal transductionpathway that indicate enzyme activity. Thus, the phosphorylation of asubstrate, activation of a protein, a change in the expression of genesthat are up- or down-regulated in response to the enzyme proteindependent signal cascade can be assayed.

Any of the biological or biochemical functions mediated by the enzymecan be used as an endpoint assay. These include all of the biochemicalor biochemical/biological events described herein, in the referencescited herein, incorporated by reference for these endpoint assaytargets, and other functions known to those of ordinary skill in the artor that can be readily identified using the information provided in theFigures, particularly FIG. 2. Specifically, a biological function of acell or tissues that expresses the enzyme can be assayed. Experimentaldata as provided in FIG. 1 indicates that the enzymes of the presentinvention are expressed in humans in teratocarcinoma and teratocarcinomaneuronal precursor cells, fetal brain, liver adenocarcinoma, lung smallcell carinoma, and the genitourinary tract, as indicated by virtualnorthern blot analysis. In addition, PCR-based tissue screening panelsindicate expression in liver.

Binding and/or activating compounds can also be screened by usingchimeric enzyme proteins in which the amino terminal extracellulardomain, or parts thereof, the entire transmembrane domain or subregions,such as any of the seven transmembrane segments or any of theintracellular or extracellular loops and the carboxy terminalintracellular domain, or parts thereof, can be replaced by heterologousdomains or subregions. For example, a substrate-binding region can beused that interacts with a different substrate then that which isrecognized by the native enzyme. Accordingly, a different set of signaltransduction components is available as an end-point assay foractivation. This allows for assays to be performed in other than thespecific host cell from which the enzyme is derived.

The proteins of the present invention are also useful in competitionbinding assays in methods designed to discover compounds that interactwith the enzyme (e.g. binding partners and/or ligands). Thus, a compoundis exposed to a enzyme polypeptide under conditions that allow thecompound to bind or to otherwise interact with the polypeptide. Solubleenzyme polypeptide is also added to the mixture. If the test compoundinteracts with the soluble enzyme polypeptide, it decreases the amountof complex formed or activity from the enzyme target. This type of assayis particularly useful in cases in which compounds are sought thatinteract with specific regions of the enzyme. Thus, the solublepolypeptide that competes with the target enzyme region is designed tocontain peptide sequences corresponding to the region of interest.

To perform cell free drug screening assays, it is sometimes desirable toimmobilize either the enzyme protein, or fragment, or its targetmolecule to facilitate separation of complexes from uncomplexed forms ofone or both of the proteins, as well as to accommodate automation of theassay.

Techniques for immobilizing proteins on matrices can be used in the drugscreening assays. In one embodiment, a fusion protein can be providedwhich adds a domain that allows the protein to be bound to a matrix. Forexample, glutathione-S-transferase fusion proteins can be adsorbed ontoglutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) orglutathione derivatized microtitre plates, which are then combined withthe cell lysates (e.g., ³⁵S-labeled) and the candidate compound, and themixture incubated under conditions conducive to complex formation (e.g.,at physiological conditions for salt and pH). Following incubation, thebeads are washed to remove any unbound label, and the matrix immobilizedand radiolabel determined directly, or in the supernatant after thecomplexes are dissociated. Alternatively, the complexes can bedissociated from the matrix, separated by SDS-PAGE, and the level ofenzyme-binding protein found in the bead fraction quantitated from thegel using standard electrophoretic techniques. For example, either thepolypeptide or its target molecule can be immobilized utilizingconjugation of biotin and streptavidin using techniques well known inthe art. Alternatively, antibodies reactive with the protein but whichdo not interfere with binding of the protein to its target molecule canbe derivatized to the wells of the plate, and the protein trapped in thewells by antibody conjugation. Preparations of a enzyme-binding proteinand a candidate compound are incubated in the enzyme protein-presentingwells and the amount of complex trapped in the well can be quantitated.Methods for detecting such complexes, in addition to those describedabove for the GST-immobilized complexes, include immunodetection ofcomplexes using antibodies reactive with the enzyme protein targetmolecule, or which are reactive with enzyme protein and compete with thetarget molecule, as well as enzyme-linked assays which rely on detectingan enzymatic activity associated with the target molecule.

Agents that modulate one of the enzymes of the present invention can beidentified using one or more of the above assays, alone or incombination. It is generally preferable to use a cell-based or cell freesystem first and then confirm activity in an animal or other modelsystem. Such model systems are well known in the art and can readily beemployed in this context.

Modulators of enzyme protein activity identified according to these drugscreening assays can be used to treat a subject with a disorder mediatedby the enzyme pathway, by treating cells or tissues that express theenzyme. Experimental data as provided in FIG. 1 indicates expression inhumans in teratocarcinoma and teratocarcinoma neuronal precursor cells,fetal brain, liver and liver adenocarcinoma, lung small cell carinoma,and the genitourinary tract. These methods of treatment include thesteps of administering a modulator of enzyme activity in apharmaceutical composition to a subject in need of such treatment, themodulator being identified as described herein.

In yet another aspect of the invention, the enzyme proteins can be usedas “bait proteins” in a two-hybrid assay or three-hybrid assay (see,e.g., U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell 72:223-232;Madura et al. (1993) J. Biol. Chem. 268:12046-12054; Bartel et al.(1993) Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene8:1693-1696; and Brent WO94/10300), to identify other proteins, whichbind to or interact with the enzyme and are involved in enzyme activity.Such enzyme-binding proteins are also likely to be involved in thepropagation of signals by the enzyme proteins or enzyme targets as, forexample, downstream elements of a enzyme-mediated signaling pathway.Alternatively, such enzyme-binding proteins are likely to be enzymeinhibitors.

The two-hybrid system is based on the modular nature of mosttranscription factors, which consist of separable DNA-binding andactivation domains. Briefly, the assay utilizes two different DNAconstructs. In one construct, the gene that codes for a enzyme proteinis fused to a gene encoding the DNA binding domain of a knowntranscription factor (e.g., GAL-4). In the other construct, a DNAsequence, from a library of DNA sequences, that encodes an unidentifiedprotein (“prey” or “sample”) is fused to a gene that codes for theactivation domain of the known transcription factor. If the “bait” andthe “prey” proteins are able to interact, in vivo, forming aenzyme-dependent complex, the DNA-binding and activation domains of thetranscription factor are brought into close proximity. This proximityallows transcription of a reporter gene (e.g., LacZ) which is operablylinked to a transcriptional regulatory site responsive to thetranscription factor. Expression of the reporter gene can be detectedand cell colonies containing the functional transcription factor can beisolated and used to obtain the cloned gene which encodes the proteinwhich interacts with the enzyme protein.

This invention further pertains to novel agents identified by theabove-described screening assays. Accordingly, it is within the scope ofthis invention to further use an agent identified as described herein inan appropriate animal model. For example, an agent identified asdescribed herein (e.g., a enzyme-modulating agent, an antisense enzymenucleic acid molecule, a enzyme-specific antibody, or a enzyme-bindingpartner) can be used in an animal or other model to determine theefficacy, toxicity, or side effects of treatment with such an agent.Alternatively, an agent identified as described herein can be used in ananimal or other model to determine the mechanism of action of such anagent. Furthermore, this invention pertains to uses of novel agentsidentified by the above-described screening assays for treatments asdescribed herein.

The enzyme proteins of the present invention are also useful to providea target for diagnosing a disease or predisposition to disease mediatedby the peptide. Accordingly, the invention provides methods fordetecting the presence, or levels of, the protein (or encoding mRNA) ina cell, tissue, or organism. Experimental data as provided in FIG. 1indicates expression in humans in teratocarcinoma and teratocarcinomaneuronal precursor cells, fetal brain, liver and liver adenocarcinoma,lung small cell carinoma, and the genitourinary tract. The methodinvolves contacting a biological sample with a compound capable ofinteracting with the enzyme protein such that the interaction can bedetected. Such an assay can be provided in a single detection format ora multi-detection format such as an antibody chip array.

One agent for detecting a protein in a sample is an antibody capable ofselectively binding to protein. A biological sample includes tissues,cells and biological fluids isolated from a subject, as well as tissues,cells and fluids present within a subject.

The peptides of the present invention also provide targets fordiagnosing active protein activity, disease, or predisposition todisease, in a patient having a variant peptide, particularly activitiesand conditions that are known for other members of the family ofproteins to which the present one belongs. Thus, the peptide can beisolated from a biological sample and assayed for the presence of agenetic mutation that results in aberrant peptide. This includes aminoacid substitution, deletion, insertion, rearrangement, (as the result ofaberrant splicing events), and inappropriate post-translationalmodification. Analytic methods include altered electrophoretic mobility,altered tryptic peptide digest, altered enzyme activity in cell-based orcell-free assay, alteration in substrate or antibody-binding pattern,altered isoelectric point, direct amino acid sequencing, and any otherof the known assay techniques useful for detecting mutations in aprotein. Such an assay can be provided in a single detection format or amulti-detection format such as an antibody chip array.

In vitro techniques for detection of peptide include enzyme linkedimmunosorbent assays (ELISAs), Western blots, immunoprecipitations andimmunofluorescence using a detection reagent, such as an antibody orprotein binding agent. Alternatively, the peptide can be detected invivo in a subject by introducing into the subject a labeled anti-peptideantibody or other types of detection agent. For example, the antibodycan be labeled with a radioactive marker whose presence and location ina subject can be detected by standard imaging techniques. Particularlyuseful are methods that detect the allelic variant of a peptideexpressed in a subject and methods which detect fragments of a peptidein a sample.

The peptides are also useful in pharmacogenomic analysis.Pharmacogenomics deal with clinically significant hereditary variationsin the response to drugs due to altered drug disposition and abnormalaction in affected persons. See, e.g., Eichelbaum, M. (Clin. Exp.Pharmacol. Physiol. 23(10-11):983-985 (1996)), and Linder, M. W. (Clin.Chem. 43(2):254-266 (1997)). The clinical outcomes of these variationsresult in severe toxicity of therapeutic drugs in certain individuals ortherapeutic failure of drugs in certain individuals as a result ofindividual variation in metabolism. Thus, the genotype of the individualcan determine the way a therapeutic compound acts on the body or the waythe body metabolizes the compound. Further, the activity of drugmetabolizing enzymes effects both the intensity and duration of drugaction. Thus, the pharmacogenomics of the individual permit theselection of effective compounds and effective dosages of such compoundsfor prophylactic or therapeutic treatment based on the individual'sgenotype. The discovery of genetic polymorphisms in some drugmetabolizing enzymes has explained why some patients do not obtain theexpected drug effects, show an exaggerated drug effect, or experienceserious toxicity from standard drug dosages. Polymorphisms can beexpressed in the phenotype of the extensive metabolizer and thephenotype of the poor metabolizer. Accordingly, genetic polymorphism maylead to allelic protein variants of the enzyme protein in which one ormore of the enzyme functions in one population is different from thosein another population. The peptides thus allow a target to ascertain agenetic predisposition that can affect treatment modality. Thus, in aligand-based treatment, polymorphism may give rise to amino terminalextracellular domains and/or other substrate-binding regions that aremore or less active in substrate binding, and enzyme activation.Accordingly, substrate dosage would necessarily be modified to maximizethe therapeutic effect within a given population containing apolymorphism. As an alternative to genotyping, specific polymorphicpeptides could be identified.

The peptides are also useful for treating a disorder characterized by anabsence of, inappropriate, or unwanted expression of the protein.Experimental data as provided in FIG. 1 indicates expression in humansin teratocarcinoma and teratocarcinoma neuronal precursor cells, fetalbrain, liver and liver adenocarcinoma, lung small cell carinoma, and thegenitourinary tract. Accordingly, methods for treatment include the useof the enzyme protein or fragments.

Antibodies

The invention also provides antibodies that selectively bind to one ofthe peptides of the present invention, a protein comprising such apeptide, as well as variants and fragments thereof. As used herein, anantibody selectively binds a target peptide when it binds the targetpeptide and does not significantly bind to unrelated proteins. Anantibody is still considered to selectively bind a peptide even if italso binds to other proteins that are not substantially homologous withthe target peptide so long as such proteins share homology with afragment or domain of the peptide target of the antibody. In this case,it would be understood that antibody binding to the peptide is stillselective despite some degree of cross-reactivity.

As used herein, an antibody is defined in terms consistent with thatrecognized within the art: they are multi-subunit proteins produced by amammalian organism in response to an antigen challenge. The antibodiesof the present invention include polyclonal antibodies and monoclonalantibodies, as well as fragments of such antibodies, including, but notlimited to, Fab or F(ab′)₂, and Fv fragments.

Many methods are known for generating and/or identifying antibodies to agiven target peptide. Several such methods are described by Harlow,Antibodies, Cold Spring Harbor Press, (1989).

In general, to generate antibodies, an isolated peptide is used as animmunogen and is administered to a mammalian organism, such as a rat,rabbit or mouse. The full-length protein, an antigenic peptide fragmentor a fusion protein can be used. Particularly important fragments arethose covering functional domains, such as the domains identified inFIG. 2, and domain of sequence homology or divergence amongst thefamily, such as those that can readily be identified using proteinalignment methods and as presented in the Figures.

Antibodies are preferably prepared from regions or discrete fragments ofthe enzyme proteins. Antibodies can be prepared from any region of thepeptide as described herein. However, preferred regions will includethose involved in function/activity and/or enzyme/binding partnerinteraction. FIG. 2 can be used to identify particularly importantregions while sequence alignment can be used to identify conserved andunique sequence fragments.

An antigenic fragment will typically comprise at least 8 contiguousamino acid residues. The antigenic peptide can comprise, however, atleast 10, 12, 14, 16 or more amino acid residues. Such fragments can beselected on a physical property, such as fragments correspond to regionsthat are located on the surface of the protein, e.g., hydrophilicregions or can be selected based on sequence uniqueness (see FIG. 2).

Detection on an antibody of the present invention can be facilitated bycoupling (i.e., physically linking) the antibody to a detectablesubstance. Examples of detectable substances include various enzymes,prosthetic groups, fluorescent materials, luminescent materials,bioluminescent materials, and radioactive materials. Examples ofsuitable enzymes include horseradish peroxidase, alkaline phosphatase,P-galactosidase, or acetylcholinesterase; examples of suitableprosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; examples ofbioluminescent materials include luciferase, luciferin, and aequorin,and examples of suitable radioactive material include ¹²⁵I, ¹³¹I, ³⁵S or³H.

Antibody Uses

The antibodies can be used to isolate one of the proteins of the presentinvention by standard techniques, such as affinity chromatography orimmunoprecipitation. The antibodies can facilitate the purification ofthe natural protein from cells and recombinantly produced proteinexpressed in host cells. In addition, such antibodies are useful todetect the presence of one of the proteins of the present invention incells or tissues to determine the pattern of expression of the proteinamong various tissues in an organism and over the course of normaldevelopment. Experimental data as provided in FIG. 1 indicates that theenzymes of the present invention are expressed in humans interatocarcinoma and teratocarcinoma neuronal precursor cells, fetalbrain, liver adenocarcinoma, lung small cell carinoma, and thegenitourinary tract, as indicated by virtual northern blot analysis. Inaddition, PCR-based tissue screening panels indicate expression inliver. Further, such antibodies can be used to detect protein in situ,in vitro, or in a cell lysate or supernatant in order to evaluate theabundance and pattern of expression. Also, such antibodies can be usedto assess abnormal tissue distribution or abnormal expression duringdevelopment or progression of a biological condition. Antibody detectionof circulating fragments of the full length protein can be used toidentify turnover.

Further, the antibodies can be used to assess expression in diseasestates such as in active stages of the disease or in an individual witha predisposition toward disease related to the protein's function. Whena disorder is caused by an inappropriate tissue distribution,developmental expression, level of expression of the protein, orexpressed/processed form, the antibody can be prepared against thenormal protein. Experimental data as provided in FIG. 1 indicatesexpression in humans in teratocarcinoma and teratocarcinoma neuronalprecursor cells, fetal brain, liver and liver adenocarcinoma, lung smallcell carinoma, and the genitourinary tract. If a disorder ischaracterized by a specific mutation in the protein, antibodies specificfor this mutant protein can be used to assay for the presence of thespecific mutant protein.

The antibodies can also be used to assess normal and aberrantsubcellular localization of cells in the various tissues in an organism.Experimental data as provided in FIG. 1 indicates expression in humansin teratocarcinoma and teratocarcinoma neuronal precursor cells, fetalbrain, liver and liver adenocarcinoma, lung small cell carinoma, and thegenitourinary tract. The diagnostic uses can be applied, not only ingenetic testing, but also in monitoring a treatment modality.Accordingly, where treatment is ultimately aimed at correctingexpression level or the presence of aberrant sequence and aberranttissue distribution or developmental expression, antibodies directedagainst the protein or relevant fragments can be used to monitortherapeutic efficacy.

Additionally, antibodies are useful in pharmacogenomic analysis. Thus,antibodies prepared against polymorphic proteins can be used to identifyindividuals that require modified treatment modalities. The antibodiesare also useful as diagnostic tools as an immunological marker foraberrant protein analyzed by electrophoretic mobility, isoelectricpoint, tryptic peptide digest, and other physical assays known to thosein the art.

The antibodies are also useful for tissue typing. Experimental data asprovided in FIG. 1 indicates expression in humans in teratocarcinoma andteratocarcinoma neuronal precursor cells, fetal brain, liver and liveradenocarcinoma, lung small cell carinoma, and the genitourinary tract.Thus, where a specific protein has been correlated with expression in aspecific tissue, antibodies that are specific for this protein can beused to identify a tissue type.

The antibodies are also useful for inhibiting protein function, forexample, blocking the binding of the enzyme peptide to a binding partnersuch as a substrate. These uses can also be applied in a therapeuticcontext in which treatment involves inhibiting the protein's function.An antibody can be used, for example, to block binding, thus modulating(agonizing or antagonizing) the peptides activity. Antibodies can beprepared against specific fragments containing sites required forfunction or against intact protein that is associated with a cell orcell membrane. See FIG. 2 for structural information relating to theproteins of the present invention.

The invention also encompasses kits for using antibodies to detect thepresence of a protein in a biological sample. The kit can compriseantibodies such as a labeled or labelable antibody and a compound oragent for detecting protein in a biological sample; means fordetermining the amount of protein in the sample; means for comparing theamount of protein in the sample with a standard; and instructions foruse. Such a kit can be supplied to detect a single protein or epitope orcan be configured to detect one of a multitude of epitopes, such as inan antibody detection array. Arrays are described in detail below fornuleic acid arrays and similar methods have been developed for antibodyarrays.

Nucleic Acid Molecules

The present invention further provides isolated nucleic acid moleculesthat encode a enzyme peptide or protein of the present invention (cDNA,transcript and genomic sequence). Such nucleic acid molecules willconsist of, consist essentially of, or comprise a nucleotide sequencethat encodes one of the enzyme peptides of the present invention, anallelic variant thereof, or an ortholog or paralog thereof.

As used herein, an “isolated” nucleic acid molecule is one that isseparated from other nucleic acid present in the natural source of thenucleic acid. Preferably, an “isolated” nucleic acid is free ofsequences which naturally flank the nucleic acid (i.e., sequenceslocated at the 5′ and 3′ ends of the nucleic acid) in the genomic DNA ofthe organism from which the nucleic acid is derived. However, there canbe some flanking nucleotide sequences, for example up to about 5 KB, 4KB, 3 KB, 2 KB, or 1 KB or less, particularly contiguous peptideencoding sequences and peptide encoding sequences within the same genebut separated by introns in the genomic sequence. The important point isthat the nucleic acid is isolated from remote and unimportant flankingsequences such that it can be subjected to the specific manipulationsdescribed herein such as recombinant expression, preparation of probesand primers, and other uses specific to the nucleic acid sequences.

Moreover, an “isolated” nucleic acid molecule, such as a transcript/cDNAmolecule, can be substantially free of other cellular material, orculture medium when produced by recombinant techniques, or chemicalprecursors or other chemicals when chemically synthesized. However, thenucleic acid molecule can be fused to other coding or regulatorysequences and still be considered isolated.

For example, recombinant DNA molecules contained in a vector areconsidered isolated. Further examples of isolated DNA molecules includerecombinant DNA molecules maintained in heterologous host cells orpurified (partially or substantially) DNA molecules in solution.Isolated RNA molecules include in vivo or in vitro RNA transcripts ofthe isolated DNA molecules of the present invention. Isolated nucleicacid molecules according to the present invention further include suchmolecules produced synthetically.

Accordingly, the present invention provides nucleic acid molecules thatconsist of the nucleotide sequence shown in FIG. 1 or 3 (SEQ ID NO:1,transcript sequence and SEQ ID NO:3, genomic sequence), or any nucleicacid molecule that encodes the protein provided in FIG. 2, SEQ ID NO:2.A nucleic acid molecule consists of a nucleotide sequence when thenucleotide sequence is the complete nucleotide sequence of the nucleicacid molecule.

The present invention further provides nucleic acid molecules thatconsist essentially of the nucleotide sequence shown in FIG. 1 or 3 (SEQID NO:1, transcript sequence and SEQ ID NO:3, genomic sequence), or anynucleic acid molecule that encodes the protein provided in FIG. 2, SEQID NO:2. A nucleic acid molecule consists essentially of a nucleotidesequence when such a nucleotide sequence is present with only a fewadditional nucleic acid residues in the final nucleic acid molecule.

The present invention further provides nucleic acid molecules thatcomprise the nucleotide sequences shown in FIG. 1 or 3 (SEQ ID NO:1,transcript sequence and SEQ ID NO:3, genomic sequence), or any nucleicacid molecule that encodes the protein provided in FIG. 2, SEQ ID NO:2.A nucleic acid molecule comprises a nucleotide sequence when thenucleotide sequence is at least part of the final nucleotide sequence ofthe nucleic acid molecule. In such a fashion, the nucleic acid moleculecan be only the nucleotide sequence or have additional nucleic acidresidues, such as nucleic acid residues that are naturally associatedwith it or heterologous nucleotide sequences. Such a nucleic acidmolecule can have a few additional nucleotides or can comprises severalhundred or more additional nucleotides. A brief description of howvarious types of these nucleic acid molecules can be readilymade/isolated is provided below.

In FIGS. 1 and 3, both coding and non-coding sequences are provided.Because of the source of the present invention, humans genomic sequence(FIG. 3) and cDNA/transcript sequences (FIG. 1), the nucleic acidmolecules in the Figures will contain genomic intronic sequences, 5′ and3′ non-coding sequences, gene regulatory regions and non-codingintergenic sequences. In general such sequence features are either notedin FIGS. 1 and 3 or can readily be identified using computational toolsknown in the art. As discussed below, some of the non-coding regions,particularly gene regulatory elements such as promoters, are useful fora variety of purposes, e.g. control of heterologous gene expression,target for identifying gene activity modulating compounds, and areparticularly claimed as fragments of the genomic sequence providedherein.

The isolated nucleic acid molecules can encode the mature protein plusadditional amino or carboxyl-terminal amino acids, or amino acidsinterior to the mature peptide (when the mature form has more than onepeptide chain, for instance). Such sequences may play a role inprocessing of a protein from precursor to a mature form, facilitateprotein trafficking, prolong or shorten protein half-life or facilitatemanipulation of a protein for assay or production, among other things.As generally is the case in situ, the additional amino acids may beprocessed away from the mature protein by cellular enzymes.

As mentioned above, the isolated nucleic acid molecules include, but arenot limited to, the sequence encoding the enzyme peptide alone, thesequence encoding the mature peptide and additional coding sequences,such as a leader or secretory sequence (e.g., a pre-pro or pro-proteinsequence), the sequence encoding the mature peptide, with or without theadditional coding sequences, plus additional non-coding sequences, forexample introns and non-coding 5′ and 3′ sequences such as transcribedbut non-translated sequences that play a role in transcription, mRNAprocessing (including splicing and polyadenylation signals), ribosomebinding and stability of mRNA. In addition, the nucleic acid moleculemay be fused to a marker sequence encoding, for example, a peptide thatfacilitates purification.

Isolated nucleic acid molecules can be in the form of RNA, such as mRNA,or in the form DNA, including cDNA and genomic DNA obtained by cloningor produced by chemical synthetic techniques or by a combinationthereof. The nucleic acid, especially DNA, can be double-stranded orsingle-stranded. Single-stranded nucleic acid can be the coding strand(sense strand) or the non-coding strand (anti-sense strand).

The invention further provides nucleic acid molecules that encodefragments of the peptides of the present invention as well as nucleicacid molecules that encode obvious variants of the enzyme proteins ofthe present invention that are described above. Such nucleic acidmolecules may be naturally occurring, such as allelic variants (samelocus), paralogs (different locus), and orthologs (different organism),or may be constructed by recombinant DNA methods or by chemicalsynthesis. Such non-naturally occurring variants may be made bymutagenesis techniques, including those applied to nucleic acidmolecules, cells, or organisms. Accordingly, as discussed above, thevariants can contain nucleotide substitutions, deletions, inversions andinsertions. Variation can occur in either or both the coding andnon-coding regions. The variations can produce both conservative andnon-conservative amino acid substitutions.

The present invention further provides non-coding fragments of thenucleic acid molecules provided in FIGS. 1 and 3. Preferred non-codingfragments include, but are not limited to, promoter sequences, enhancersequences, gene modulating sequences and gene termination sequences.Such fragments are useful in controlling heterologous gene expressionand in developing screens to identify gene-modulating agents. A promotercan readily be identified as being 5′ to the ATG start site in thegenomic sequence provided in FIG. 3.

A fragment comprises a contiguous nucleotide sequence greater than 12 ormore nucleotides. Further, a fragment could at least 30, 40, 50, 100,250 or 500 nucleotides in length. The length of the fragment will bebased on its intended use. For example, the fragment can encode epitopebearing regions of the peptide, or can be useful as DNA probes andprimers. Such fragments can be isolated using the known nucleotidesequence to synthesize an oligonucleotide probe. A labeled probe canthen be used to screen a cDNA library, genomic DNA library, or mRNA toisolate nucleic acid corresponding to the coding region. Further,primers can be used in PCR reactions to clone specific regions of gene.

A probe/primer typically comprises substantially a purifiedoligonucleotide or oligonucleotide pair. The oligonucleotide typicallycomprises a region of nucleotide sequence that hybridizes understringent conditions to at least about 12, 20, 25, 40, 50 or moreconsecutive nucleotides.

Orthologs, homologs, and allelic variants can be identified usingmethods well known in the art. As described in the Peptide Section,these variants comprise a nucleotide sequence encoding a peptide that istypically 60-70%, 70-80%, 80-90%, and more typically at least about90-95% or more homologous to the nucleotide sequence shown in the Figuresheets or a fragment of this sequence. Such nucleic acid molecules canreadily be identified as being able to hybridize under moderate tostringent conditions, to the nucleotide sequence shown in the Figuresheets or a fragment of the sequence. Allelic variants can readily bedetermined by genetic locus of the encoding gene. The gene encoding thenovel enzyme of the present invention is located on a genome componentthat has been mapped to human chromosome 5 (as indicated in FIG. 3),which is supported by multiple lines of evidence, such as STS and BACmap data.

FIG. 3 provides information on SNPs that have been found in the geneencoding the enzyme of the present invention. SNPs were identified at 16different nucleotide positions. Some of these SNPs that are locatedoutside the ORF and in introns may affect gene transcription.

As used herein, the term “hybridizes under stringent conditions” isintended to describe conditions for hybridization and washing underwhich nucleotide sequences encoding a peptide at least 60-70% homologousto each other typically remain hybridized to each other. The conditionscan be such that sequences at least about 60%, at least about 70%, or atleast about 80% or more homologous to each other typically remainhybridized to each other. Such stringent conditions are known to thoseskilled in the art and can be found in Current Protocols in MolecularBiology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. One example ofstringent hybridization conditions are hybridization in 6×sodiumchloride/sodium citrate (SSC) at about 45C, followed by one or morewashes in 0.2×SSC, 0.1% SDS at 50-65 C. Examples of moderate to lowstringency hybridization conditions are well known in the art.

Nucleic Acid Molecule Uses

The nucleic acid molecules of the present invention are useful forprobes, primers, chemical intermediates, and in biological assays. Thenucleic acid molecules are useful as a hybridization probe for messengerRNA, transcript/cDNA and genomic DNA to isolate full-length cDNA andgenomic clones encoding the peptide described in FIG. 2 and to isolatecDNA and genomic clones that correspond to variants (alleles, orthologs,etc.) producing the same or related peptides shown in FIG. 2. Asillustrated in FIG. 3, SNPs were identified at 16 different nucleotidepositions.

The probe can correspond to any sequence along the entire length of thenucleic acid molecules provided in the Figures. Accordingly, it could bederived from 5′ noncoding regions, the coding region, and 3′ noncodingregions. However, as discussed, fragments are not to be construed asencompassing fragments disclosed prior to the present invention.

The nucleic acid molecules are also useful as primers for PCR to amplifyany given region of a nucleic acid molecule and are useful to synthesizeantisense molecules of desired length and sequence.

The nucleic acid molecules are also useful for constructing recombinantvectors. Such vectors include expression vectors that express a portionof, or all of, the peptide sequences. Vectors also include insertionvectors, used to integrate into another nucleic acid molecule sequence,such as into the cellular genome, to alter in situ expression of a geneand/or gene product. For example, an endogenous coding sequence can bereplaced via homologous recombination with all or part of the codingregion containing one or more specifically introduced mutations.

The nucleic acid molecules are also useful for expressing antigenicportions of the proteins.

The nucleic acid molecules are also useful as probes for determining thechromosomal positions of the nucleic acid molecules by means of in situhybridization methods. The gene encoding the novel enzyme of the presentinvention is located on a genome component that has been mapped to humanchromosome 5 (as indicated in FIG. 3), which is supported by multiplelines of evidence, such as STS and BAC map data.

The nucleic acid molecules are also useful in making vectors containingthe gene regulatory regions of the nucleic acid molecules of the presentinvention.

The nucleic acid molecules are also useful for designing ribozymescorresponding to all, or a part, of the mRNA produced from the nucleicacid molecules described herein.

The nucleic acid molecules are also useful for making vectors thatexpress part, or all, of the peptides.

The nucleic acid molecules are also useful for constructing host cellsexpressing a part, or all, of the nucleic acid molecules and peptides.

The nucleic acid molecules are also useful for constructing transgenicanimals expressing all, or a part, of the nucleic acid molecules andpeptides.

The nucleic acid molecules are also useful as hybridization probes fordetermining the presence, level, form and distribution of nucleic acidexpression. Experimental data as provided in FIG. 1 indicates that theenzymes of the present invention are expressed in humans interatocarcinoma and teratocarcinoma neuronal precursor cells, fetalbrain, liver adenocarcinoma, lung small cell carinoma, and thegenitourinary tract, as indicated by virtual northern blot analysis. Inaddition, PCR-based tissue screening panels indicate expression inliver. Accordingly, the probes can be used to detect the presence of, orto determine levels of, a specific nucleic acid molecule in cells,tissues, and in organisms. The nucleic acid whose level is determinedcan be DNA or RNA. Accordingly, probes corresponding to the peptidesdescribed herein can be used to assess expression and/or gene copynumber in a given cell, tissue, or organism. These uses are relevant fordiagnosis of disorders involving an increase or decrease in enzymeprotein expression relative to normal results.

In vitro techniques for detection of mRNA include Northernhybridizations and in situ hybridizations. In vitro techniques fordetecting DNA includes Southern hybridizations and in situhybridization.

Probes can be used as a part of a diagnostic test kit for identifyingcells or tissues that express a enzyme protein, such as by measuring alevel of a enzyme-encoding nucleic acid in a sample of cells from asubject e.g., mRNA or genomic DNA, or determining if a enzyme gene hasbeen mutated. Experimental data as provided in FIG. 1 indicates that theenzymes of the present invention are expressed in humans interatocarcinoma and teratocarcinoma neuronal precursor cells, fetalbrain, liver adenocarcinoma, lung small cell carinoma, and thegenitourinary tract, as indicated by virtual northern blot analysis. Inaddition, PCR-based tissue screening panels indicate expression inliver.

Nucleic acid expression assays are useful for drug screening to identifycompounds that modulate enzyme nucleic acid expression.

The invention thus provides a method for identifying a compound that canbe used to treat a disorder associated with nucleic acid expression ofthe enzyme gene, particularly biological and pathological processes thatare mediated by the enzyme in cells and tissues that express it.Experimental data as provided in FIG. 1 indicates expression in humansin teratocarcinoma and teratocarcinoma neuronal precursor cells, fetalbrain, liver and liver adenocarcinoma, lung small cell carinoma, and thegenitourinary tract. The method typically includes assaying the abilityof the compound to modulate the expression of the enzyme nucleic acidand thus identifying a compound that can be used to treat a disordercharacterized by undesired enzyme nucleic acid expression. The assayscan be performed in cell-based and cell-free systems. Cell-based assaysinclude cells naturally expressing the enzyme nucleic acid orrecombinant cells genetically engineered to express specific nucleicacid sequences.

The assay for enzyme nucleic acid expression can involve direct assay ofnucleic acid levels, such as mRNA levels, or on collateral compoundsinvolved in the signal pathway. Further, the expression of genes thatare up- or down-regulated in response to the enzyme protein signalpathway can also be assayed. In this embodiment the regulatory regionsof these genes can be operably linked to a reporter gene such asluciferase.

Thus, modulators of enzyme gene expression can be identified in a methodwherein a cell is contacted with a candidate compound and the expressionof mRNA determined. The level of expression of enzyme mRNA in thepresence of the candidate compound is compared to the level ofexpression of enzyme mRNA in the absence of the candidate compound. Thecandidate compound can then be identified as a modulator of nucleic acidexpression based on this comparison and be used, for example to treat adisorder characterized by aberrant nucleic acid expression. Whenexpression of mRNA is statistically significantly greater in thepresence of the candidate compound than in its absence, the candidatecompound is identified as a stimulator of nucleic acid expression. Whennucleic acid expression is statistically significantly less in thepresence of the candidate compound than in its absence, the candidatecompound is identified as an inhibitor of nucleic acid expression.

The invention further provides methods of treatment, with the nucleicacid as a target, using a compound identified through drug screening asa gene modulator to modulate enzyme nucleic acid expression in cells andtissues that express the enzyme. Experimental data as provided in FIG. 1indicates that the enzymes of the present invention are expressed inhumans in teratocarcinoma and teratocarcinoma neuronal precursor cells,fetal brain, liver adenocarcinoma, lung small cell carinoma, and thegenitourinary tract, as indicated by virtual northern blot analysis. Inaddition, PCR-based tissue screening panels indicate expression inliver. Modulation includes both up-regulation (i.e. activation oragonization) or down-regulation (suppression or antagonization) ornucleic acid expression.

Alternatively, a modulator for enzyme nucleic acid expression can be asmall molecule or drug identified using the screening assays describedherein as long as the drug or small molecule inhibits the enzyme nucleicacid expression in the cells and tissues that express the protein.Experimental data as provided in FIG. 1 indicates expression in humansin teratocarcinoma and teratocarcinoma neuronal precursor cells, fetalbrain, liver and liver adenocarcinoma, lung small cell carinoma, and thegenitourinary tract.

The nucleic acid molecules are also useful for monitoring theeffectiveness of modulating compounds on the expression or activity ofthe enzyme gene in clinical trials or in a treatment regimen. Thus, thegene expression pattern can serve as a barometer for the continuingeffectiveness of treatment with the compound, particularly withcompounds to which a patient can develop resistance. The gene expressionpattern can also serve as a marker indicative of a physiologicalresponse of the affected cells to the compound. Accordingly, suchmonitoring would allow either increased administration of the compoundor the administration of alternative compounds to which the patient hasnot become resistant. Similarly, if the level of nucleic acid expressionfalls below a desirable level, administration of the compound could becommensurately decreased.

The nucleic acid molecules are also useful in diagnostic assays forqualitative changes in enzyme nucleic acid expression, and particularlyin qualitative changes that lead to pathology. The nucleic acidmolecules can be used to detect mutations in enzyme genes and geneexpression products such as mRNA. The nucleic acid molecules can be usedas hybridization probes to detect naturally occurring genetic mutationsin the enzyme gene and thereby to determine whether a subject with themutation is at risk for a disorder caused by the mutation. Mutationsinclude deletion, addition, or substitution of one or more nucleotidesin the gene, chromosomal rearrangement, such as inversion ortransposition, modification of genomic DNA, such as aberrant methylationpatterns or changes in gene copy number, such as amplification.Detection of a mutated form of the enzyme gene associated with adysfunction provides a diagnostic tool for an active disease orsusceptibility to disease when the disease results from overexpression,underexpression, or altered expression of a enzyme protein.

Individuals carrying mutations in the enzyme gene can be detected at thenucleic acid level by a variety of techniques. FIG. 3 providesinformation on SNPs that have been found in the gene encoding the enzymeof the present invention. SNPs were identified at 16 differentnucleotide positions. Some of these SNPs that are located outside theORF and in introns may affect gene transcription. The gene encoding thenovel enzyme of the present invention is located on a genome componentthat has been mapped to human chromosome 5 (as indicated in FIG. 3),which is supported by multiple lines of evidence, such as STS and BACmap data. Genomic DNA can be analyzed directly or can be amplified byusing PCR prior to analysis. RNA or cDNA can be used in the same way. Insome uses, detection of the mutation involves the use of a probe/primerin a polymerase chain reaction (PCR) (see, e.g. U.S. Pat. Nos. 4,683,195and 4,683,202), such as anchor PCR or RACE PCR, or, alternatively, in aligation chain reaction (LCR) (see, e.g., Landegran et al., Science241:1077-1080 (1988); and Nakazawa et al., PNAS 91:360-364 (1994)), thelatter of which can be particularly useful for detecting point mutationsin the gene (see Abravaya et al., Nucleic Acids Res. 23:675-682 (1995)).This method can include the steps of collecting a sample of cells from apatient, isolating nucleic acid (e.g., genomic, mRNA or both) from thecells of the sample, contacting the nucleic acid sample with one or moreprimers which specifically hybridize to a gene under conditions suchthat hybridization and amplification of the gene (if present) occurs,and detecting the presence or absence of an amplification product, ordetecting the size of the amplification product and comparing the lengthto a control sample. Deletions and insertions can be detected by achange in size of the amplified product compared to the normal genotype.Point mutations can be identified by hybridizing amplified DNA to normalRNA or antisense DNA sequences.

Alternatively, mutations in a enzyme gene can be directly identified,for example, by alterations in restriction enzyme digestion patternsdetermined by gel electrophoresis.

Further, sequence-specific ribozymes (U.S. Pat. No. 5,498,531) can beused to score for the presence of specific mutations by development orloss of a ribozyme cleavage site. Perfectly matched sequences can bedistinguished from mismatched sequences by nuclease cleavage digestionassays or by differences in melting temperature.

Sequence changes at specific locations can also be assessed by nucleaseprotection assays such as RNase and S1 protection or the chemicalcleavage method. Furthermore, sequence differences between a mutantenzyme gene and a wild-type gene can be determined by direct DNAsequencing. A variety of automated sequencing procedures can be utilizedwhen performing the diagnostic assays (Naeve, C. W., (1995)Biotechniques 19:448), including sequencing by mass spectrometry (see,e.g., PCT International Publication No. WO 94/16101; Cohen et al., Adv.Chromatogr. 36:127-162 (1996); and Griffin et al., Appl. Biochem.Biotechnol. 38:147-159 (1993)).

Other methods for detecting mutations in the gene include methods inwhich protection from cleavage agents is used to detect mismatched basesin RNA/RNA or RNA/DNA duplexes (Myers et al., Science 230:1242 (1985));Cotton et al, PNAS 85:4397 (1988); Saleeba et al., Meth. Enzymol.217:286-295 (1992)), electrophoretic mobility of mutant and wild typenucleic acid is compared (Orita et al., PNAS 86:2766 (1989); Cotton etal., Mutat. Res. 285:125-144 (1993); and Hayashi et al., Genet. Anal.Tech. Appl. 9:73-79 (1992)), and movement of mutant or wild-typefragments in polyacrylamide gels containing a gradient of denaturant isassayed using denaturing gradient gel electrophoresis (Myers et al.,Nature 313:495 (1985)). Examples of other techniques for detecting pointmutations include selective oligonucleotide hybridization, selectiveamplification, and selective primer extension.

The nucleic acid molecules are also useful for testing an individual fora genotype that while not necessarily causing the disease, neverthelessaffects the treatment modality. Thus, the nucleic acid molecules can beused to study the relationship between an individual's genotype and theindividual's response to a compound used for treatment (pharmacogenomicrelationship). Accordingly, the nucleic acid molecules described hereincan be used to assess the mutation content of the enzyme gene in anindividual in order to select an appropriate compound or dosage regimenfor treatment. FIG. 3 provides information on SNPs that have been foundin the gene encoding the enzyme of the present invention. SNPs wereidentified at 16 different nucleotide positions. Some of these SNPs thatare located outside the ORF and in introns may affect genetranscription.

Thus nucleic acid molecules displaying genetic variations that affecttreatment provide a diagnostic target that can be used to tailortreatment in an individual. Accordingly, the production of recombinantcells and animals containing these polymorphisms allow effectiveclinical design of treatment compounds and dosage regimens.

The nucleic acid molecules are thus useful as antisense constructs tocontrol enzyme gene expression in cells, tissues, and organisms. A DNAantisense nucleic acid molecule is designed to be complementary to aregion of the gene involved in transcription, preventing transcriptionand hence production of enzyme protein. An antisense RNA or DNA nucleicacid molecule would hybridize to the mRNA and thus block translation ofmRNA into enzyme protein.

Alternatively, a class of antisense molecules can be used to inactivatemRNA in order to decrease expression of enzyme nucleic acid.Accordingly, these molecules can treat a disorder characterized byabnormal or undesired enzyme nucleic acid expression. This techniqueinvolves cleavage by means of ribozymes containing nucleotide sequencescomplementary to one or more regions in the mRNA that attenuate theability of the mRNA to be translated. Possible regions include codingregions and particularly coding regions corresponding to the catalyticand other functional activities of the enzyme protein, such as substratebinding.

The nucleic acid molecules also provide vectors for gene therapy inpatients containing cells that are aberrant in enzyme gene expression.Thus, recombinant cells, which include the patient's cells that havebeen engineered ex vivo and returned to the patient, are introduced intoan individual where the cells produce the desired enzyme protein totreat the individual.

The invention also encompasses kits for detecting the presence of aenzyme nucleic acid in a biological sample. Experimental data asprovided in FIG. 1 indicates that the enzymes of the present inventionare expressed in humans in teratocarcinoma and teratocarcinoma neuronalprecursor cells, fetal brain, liver adenocarcinoma, lung small cellcarinoma, and the genitourinary tract, as indicated by virtual northernblot analysis. In addition, PCR-based tissue screening panels indicateexpression in liver. For example, the kit can comprise reagents such asa labeled or labelable nucleic acid or agent capable of detecting enzymenucleic acid in a biological sample; means for determining the amount ofenzyme nucleic acid in the sample; and means for comparing the amount ofenzyme nucleic acid in the sample with a standard. The compound or agentcan be packaged in a suitable container. The kit can further compriseinstructions for using the kit to detect enzyme protein mRNA or DNA.

Nucleic Acid Arrays

The present invention further provides nucleic acid detection kits, suchas arrays or microarrays of nucleic acid molecules that are based on thesequence information provided in FIGS. 1 and 3 (SEQ ID NOS:1 and 3).

As used herein “Arrays” or “Microarrays” refers to an array of distinctpolynucleotides or oligonucleotides synthesized on a substrate, such aspaper, nylon or other type of membrane, filter, chip, glass slide, orany other suitable solid support. In one embodiment, the microarray isprepared and used according to the methods described in U.S. Pat. No.5,837,832, Chee et al., PCT application WO95/11995 (Chee et al.),Lockhart, D. J. et al. (1996; Nat. Biotech. 14: 1675-1680) and Schena,M. et al. (1996; Proc. Natl. Acad. Sci. 93: 10614-10619), all of whichare incorporated herein in their entirety by reference. In otherembodiments, such arrays are produced by the methods described by Brownet al., U.S. Pat. No. 5,807,522.

The microarray or detection kit is preferably composed of a large numberof unique, single-stranded nucleic acid sequences, usually eithersynthetic antisense oligonucleotides or fragments of cDNAs, fixed to asolid support. The oligonucleotides are preferably about 6-60nucleotides in length, more preferably 15-30 nucleotides in length, andmost preferably about 20-25 nucleotides in length. For a certain type ofmicroarray or detection kit, it may be preferable to useoligonucleotides that are only 7-20 nucleotides in length. Themicroarray or detection kit may contain oligonucleotides that cover theknown 5′, or 3′, sequence, sequential oligonucleotides which cover thefull length sequence; or unique oligonucleotides selected fromparticular areas along the length of the sequence. Polynucleotides usedin the microarray or detection kit may be oligonucleotides that arespecific to a gene or genes of interest.

In order to produce oligonucleotides to a known sequence for amicroarray or detection kit, the gene(s) of interest (or an ORFidentified from the contigs of the present invention) is typicallyexamined using a computer algorithm which starts at the 5′ or at the 3′end of the nucleotide sequence. Typical algorithms will then identifyoligomers of defined length that are unique to the gene, have a GCcontent within a range suitable for hybridization, and lack predictedsecondary structure that may interfere with hybridization. In certainsituations it may be appropriate to use pairs of oligonucleotides on amicroarray or detection kit. The “pairs” will be identical, except forone nucleotide that preferably is located in the center of the sequence.The second oligonucleotide in the pair (mismatched by one) serves as acontrol. The number of oligonucleotide pairs may range from two to onemillion. The oligomers are synthesized at designated areas on asubstrate using a light-directed chemical process. The substrate may bepaper, nylon or other type of membrane, filter, chip, glass slide or anyother suitable solid support.

In another aspect, an oligonucleotide may be synthesized on the surfaceof the substrate by using a chemical coupling procedure and an ink jetapplication apparatus, as described in PCT application WO95/251116(Baldeschweiler et al.) which is incorporated herein in its entirety byreference. In another aspect, a “gridded” array analogous to a dot (orslot) blot may be used to arrange and link cDNA fragments oroligonucleotides to the surface of a substrate using a vacuum system,thermal, UV, mechanical or chemical bonding procedures. An array, suchas those described above, may be produced by hand or by using availabledevices (slot blot or dot blot apparatus), materials (any suitable solidsupport), and machines (including robotic instruments), and may contain8, 24, 96, 384, 1536, 6144 or more oligonucleotides, or any other numberbetween two and one million which lends itself to the efficient use ofcommercially available instrumentation.

In order to conduct sample analysis using a microarray or detection kit,the RNA or DNA from a biological sample is made into hybridizationprobes. The mRNA is isolated, and cDNA is produced and used as atemplate to make antisense RNA (aRNA). The aRNA is amplified in thepresence of fluorescent nucleotides, and labeled probes are incubatedwith the microarray or detection kit so that the probe sequenceshybridize to complementary oligonucleotides of the microarray ordetection kit. Incubation conditions are adjusted so that hybridizationoccurs with precise complementary matches or with various degrees ofless complementarity. After removal of nonhybridized probes, a scanneris used to determine the levels and patterns of fluorescence. Thescanned images are examined to determine degree of complementarity andthe relative abundance of each oligonucleotide sequence on themicroarray or detection kit. The biological samples may be obtained fromany bodily fluids (such as blood, urine, saliva, phlegm, gastric juices,etc.), cultured cells, biopsies, or other tissue preparations. Adetection system may be used to measure the absence, presence, andamount of hybridization for all of the distinct sequencessimultaneously. This data may be used for large-scale correlationstudies on the sequences, expression patterns, mutations, variants, orpolymorphisms among samples.

Using such arrays, the present invention provides methods to identifythe expression of the enzyme proteins/peptides of the present invention.In detail, such methods comprise incubating a test sample with one ormore nucleic acid molecules and assaying for binding of the nucleic acidmolecule with components within the test sample. Such assays willtypically involve arrays comprising many genes, at least one of which isa gene of the present invention and or alleles of the enzyme gene of thepresent invention. FIG. 3 provides information on SNPs that have beenfound in the gene encoding the enzyme of the present invention. SNPswere identified at 16 different nucleotide positions. Some of these SNPsthat are located outside the ORF and in introns may affect genetranscription.

Conditions for incubating a nucleic acid molecule with a test samplevary. Incubation conditions depend on the format employed in the assay,the detection methods employed, and the type and nature of the nucleicacid molecule used in the assay. One skilled in the art will recognizethat any one of the commonly available hybridization, amplification orarray assay formats can readily be adapted to employ the novel fragmentsof the Human genome disclosed herein. Examples of such assays can befound in Chard, T, An Introduction to Radioimmunoassay and RelatedTechniques, Elsevier Science Publishers, Amsterdam, The Netherlands(1986); Bullock, G. R. et al., Techniques in Immunocytochemistry,Academic Press, Orlando, Fla. Vol. 1 (1982), Vol. 2 (1983), Vol. 3(1985); Tijssen, P., Practice and Theory of Enzyme Immunoassays:Laboratory Techniques in Biochemistry and Molecular Biology, ElsevierScience Publishers, Amsterdam, The Netherlands (1985).

The test samples of the present invention include cells, protein ormembrane extracts of cells. The test sample used in the above-describedmethod will vary based on the assay format, nature of the detectionmethod and the tissues, cells or extracts used as the sample to beassayed. Methods for preparing nucleic acid extracts or of cells arewell known in the art and can be readily be adapted in order to obtain asample that is compatible with the system utilized.

In another embodiment of the present invention, kits are provided whichcontain the necessary reagents to carry out the assays of the presentinvention.

Specifically, the invention provides a compartmentalized kit to receive,in close confinement, one or more containers which comprises: (a) afirst container comprising one of the nucleic acid molecules that canbind to a fragment of the Human genome disclosed herein; and (b) one ormore other containers comprising one or more of the following: washreagents, reagents capable of detecting presence of a bound nucleicacid.

In detail, a compartmentalized kit includes any kit in which reagentsare contained in separate containers. Such containers include smallglass containers, plastic containers, strips of plastic, glass or paper,or arraying material such as silica. Such containers allows one toefficiently transfer reagents from one compartment to anothercompartment such that the samples and reagents are notcross-contaminated, and the agents or solutions of each container can beadded in a quantitative fashion from one compartment to another. Suchcontainers will include a container which will accept the test sample, acontainer which contains the nucleic acid probe, containers whichcontain wash reagents (such as phosphate buffered saline, Tris-buffers,etc.), and containers which contain the reagents used to detect thebound probe. One skilled in the art will readily recognize that thepreviously unidentified enzyme gene of the present invention can beroutinely identified using the sequence information disclosed herein canbe readily incorporated into one of the established kit formats whichare well known in the art, particularly expression arrays.

Vectors/Host Cells

The invention also provides vectors containing the nucleic acidmolecules described herein. The term “vector” refers to a vehicle,preferably a nucleic acid molecule, which can transport the nucleic acidmolecules. When the vector is a nucleic acid molecule, the nucleic acidmolecules are covalently linked to the vector nucleic acid. With thisaspect of the invention, the vector includes a plasmid, single or doublestranded phage, a single or double stranded RNA or DNA viral vector, orartificial chromosome, such as a BAC, PAC, YAC, OR MAC.

A vector can be maintained in the host cell as an extrachromosomalelement where it replicates and produces additional copies of thenucleic acid molecules. Alternatively, the vector may integrate into thehost cell genome and produce additional copies of the nucleic acidmolecules when the host cell replicates.

The invention provides vectors for the maintenance (cloning vectors) orvectors for expression (expression vectors) of the nucleic acidmolecules. The vectors can function in prokaryotic or eukaryotic cellsor in both (shuttle vectors).

Expression vectors contain cis-acting regulatory regions that areoperably linked in the vector to the nucleic acid molecules such thattranscription of the nucleic acid molecules is allowed in a host cell.The nucleic acid molecules can be introduced into the host cell with aseparate nucleic acid molecule capable of affecting transcription. Thus,the second nucleic acid molecule may provide a trans-acting factorinteracting with the cis-regulatory control region to allowtranscription of the nucleic acid molecules from the vector.Alternatively, a trans-acting factor may be supplied by the host cell.Finally, a trans-acting factor can be produced from the vector itself.It is understood, however, that in some embodiments, transcriptionand/or translation of the nucleic acid molecules can occur in acell-free system.

The regulatory sequence to which the nucleic acid molecules describedherein can be operably linked include promoters for directing mRNAtranscription. These include, but are not limited to, the left promoterfrom bacteriophage λ, the lac, TRP, and TAC promoters from E. coli, theearly and late promoters from SV40, the CMV immediate early promoter,the adenovirus early and late promoters, and retrovirus long-terminalrepeats.

In addition to control regions that promote transcription, expressionvectors may also include regions that modulate transcription, such asrepressor binding sites and enhancers. Examples include the SV40enhancer, the cytomegalovirus immediate early enhancer, polyomaenhancer, adenovirus enhancers, and retrovirus LTR enhancers.

In addition to containing sites for transcription initiation andcontrol, expression vectors can also contain sequences necessary fortranscription termination and, in the transcribed region a ribosomebinding site for translation. Other regulatory control elements forexpression include initiation and termination codons as well aspolyadenylation signals. The person of ordinary skill in the art wouldbe aware of the numerous regulatory sequences that are useful inexpression vectors. Such regulatory sequences are described, forexample, in Sambrook et al., Molecular Cloning: A Laboratory Manual.2nd. ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,(1989).

A variety of expression vectors can be used to express a nucleic acidmolecule. Such vectors include chromosomal, episomal, and virus-derivedvectors, for example vectors derived from bacterial plasmids, frombacteriophage, from yeast episomes, from yeast chromosomal elements,including yeast artificial chromosomes, from viruses such asbaculoviruses, papovaviruses such as SV40, Vaccinia viruses,adenoviruses, poxviruses, pseudorabies viruses, and retroviruses.Vectors may also be derived from combinations of these sources such asthose derived from plasmid and bacteriophage genetic elements, e.g.cosmids and phagemids. Appropriate cloning and expression vectors forprokaryotic and eukaryotic hosts are described in Sambrook et al.,Molecular Cloning: A Laboratory Manual. 2nd. ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., (1989).

The regulatory sequence may provide constitutive expression in one ormore host cells (i.e. tissue specific) or may provide for inducibleexpression in one or more cell types such as by temperature, nutrientadditive, or exogenous factor such as a hormone or other ligand. Avariety of vectors providing for constitutive and inducible expressionin prokaryotic and eukaryotic hosts are well known to those of ordinaryskill in the art.

The nucleic acid molecules can be inserted into the vector nucleic acidby well-known methodology. Generally, the DNA sequence that willultimately be expressed is joined to an expression vector by cleavingthe DNA sequence and the expression vector with one or more restrictionenzymes and then ligating the fragments together. Procedures forrestriction enzyme digestion and ligation are well known to those ofordinary skill in the art.

The vector containing the appropriate nucleic acid molecule can beintroduced into an appropriate host cell for propagation or expressionusing well-known techniques. Bacterial cells include, but are notlimited to, E. coli, Streptomyces, and Salmonella typhimurium.Eukaryotic cells include, but are not limited to, yeast, insect cellssuch as Drosophila, animal cells such as COS and CHO cells, and plantcells.

As described herein, it may be desirable to express the peptide as afusion protein. Accordingly, the invention provides fusion vectors thatallow for the production of the peptides. Fusion vectors can increasethe expression of a recombinant protein, increase the solubility of therecombinant protein, and aid in the purification of the protein byacting for example as a ligand for affinity purification. A proteolyticcleavage site may be introduced at the junction of the fusion moiety sothat the desired peptide can ultimately be separated from the fusionmoiety. Proteolytic enzymes include, but are not limited to, factor Xa,thrombin, and enteroenzyme. Typical fusion expression vectors includepGEX (Smith et al., Gene 67:31-40 (1988)), pMAL (New England Biolabs,Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuseglutathione S-transferase (GST), maltose E binding protein, or proteinA, respectively, to the target recombinant protein. Examples of suitableinducible non-fusion E. coli expression vectors include pTrc (Amann etal., Gene 69:301-315 (1988)) and pET 11d (Studier et al., GeneExpression Technology: Methods in Enzymology 185:60-89 (1990)).

Recombinant protein expression can be maximized in host bacteria byproviding a genetic background wherein the host cell has an impairedcapacity to proteolytically cleave the recombinant protein. (Gottesman,S., Gene Expression Technology: Methods in Enzymology 185, AcademicPress, San Diego, Calif. (1990) 119-128). Alternatively, the sequence ofthe nucleic acid molecule of interest can be altered to providepreferential codon usage for a specific host cell, for example E. coli.(Wada et al., Nucleic Acids Res. 20:2111-2118 (1992)).

The nucleic acid molecules can also be expressed by expression vectorsthat are operative in yeast. Examples of vectors for expression in yeaste.g., S. cerevisiae include pYepSec1 (Baldari, et al., EMBO J 6:229-234(1987)), pMFa (Kurjan et al., Cell 30:933-943(1982)), pJRY88 (Schultz etal., Gene 54:113-123 (1987)), and pYES2 (Invitrogen Corporation, SanDiego, Calif.).

The nucleic acid molecules can also be expressed in insect cells using,for example, baculovirus expression vectors. Baculovirus vectorsavailable for expression of proteins in cultured insect cells (e.g., Sf9 cells) include the pAc series (Smith et al., Mol. Cell Biol.3:2156-2165 (1983)) and the pVL series (Lucklow et al., Virology170:31-39 (1989)).

In certain embodiments of the invention, the nucleic acid moleculesdescribed herein are expressed in mammalian cells using mammalianexpression vectors. Examples of mammalian expression vectors includepCDM8 (Seed, B. Nature 329:840(1987)) and pMT2PC (Kaufman et al., EMBO J6:187-195 (1987)).

The expression vectors listed herein are provided by way of example onlyof the well-known vectors available to those of ordinary skill in theart that would be useful to express the nucleic acid molecules. Theperson of ordinary skill in the art would be aware of other vectorssuitable for maintenance propagation or expression of the nucleic acidmolecules described herein. These are found for example in Sambrook, J.,Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual.2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989.

The invention also encompasses vectors in which the nucleic acidsequences described herein are cloned into the vector in reverseorientation, but operably linked to a regulatory sequence that permitstranscription of antisense RNA. Thus, an antisense transcript can beproduced to all, or to a portion, of the nucleic acid molecule sequencesdescribed herein, including both coding and non-coding regions.Expression of this antisense RNA is subject to each of the parametersdescribed above in relation to expression of the sense RNA (regulatorysequences, constitutive or inducible expression, tissue-specificexpression).

The invention also relates to recombinant host cells containing thevectors described herein. Host cells therefore include prokaryoticcells, lower eukaryotic cells such as yeast, other eukaryotic cells suchas insect cells, and higher eukaryotic cells such as mammalian cells.

The recombinant host cells are prepared by introducing the vectorconstructs described herein into the cells by techniques readilyavailable to the person of ordinary skill in the art. These include, butare not limited to, calcium phosphate transfection,DEAE-dextran-mediated transfection, cationic lipid-mediatedtransfection, electroporation, transduction, infection, lipofection, andother techniques such as those found in Sambrook, et al. (MolecularCloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).

Host cells can contain more than one vector. Thus, different nucleotidesequences can be introduced on different vectors of the same cell.Similarly, the nucleic acid molecules can be introduced either alone orwith other nucleic acid molecules that are not related to the nucleicacid molecules such as those providing trans-acting factors forexpression vectors. When more than one vector is introduced into a cell,the vectors can be introduced independently, co-introduced or joined tothe nucleic acid molecule vector.

In the case of bacteriophage and viral vectors, these can be introducedinto cells as packaged or encapsulated virus by standard procedures forinfection and transduction. Viral vectors can be replication-competentor replication-defective. In the case in which viral replication isdefective, replication will occur in host cells providing functions thatcomplement the defects.

Vectors generally include selectable markers that enable the selectionof the subpopulation of cells that contain the recombinant vectorconstructs. The marker can be contained in the same vector that containsthe nucleic acid molecules described herein or may be on a separatevector. Markers include tetracycline or ampicillin-resistance genes forprokaryotic host cells and dihydrofolate reductase or neomycinresistance for eukaryotic host cells. However, any marker that providesselection for a phenotypic trait will be effective.

While the mature proteins can be produced in bacteria, yeast, mammaliancells, and other cells under the control of the appropriate regulatorysequences, cell-free transcription and translation systems can also beused to produce these proteins using RNA derived from the DNA constructsdescribed herein.

Where secretion of the peptide is desired, which is difficult to achievewith multi-transmembrane domain containing proteins such as enzymes,appropriate secretion signals are incorporated into the vector. Thesignal sequence can be endogenous to the peptides or heterologous tothese peptides.

Where the peptide is not secreted into the medium, which is typicallythe case with enzymes, the protein can be isolated from the host cell bystandard disruption procedures, including freeze thaw, sonication,mechanical disruption, use of lysing agents and the like. The peptidecan then be recovered and purified by well-known purification methodsincluding ammonium sulfate precipitation, acid extraction, anion orcationic exchange chromatography, phosphocellulose chromatography,hydrophobic-interaction chromatography, affinity chromatography,hydroxylapatite chromatography, lectin chromatography, or highperformance liquid chromatography.

It is also understood that depending upon the host cell in recombinantproduction of the peptides described herein, the peptides can havevarious glycosylation patterns, depending upon the cell, or maybenon-glycosylated as when produced in bacteria. In addition, the peptidesmay include an initial modified methionine in some cases as a result ofa host-mediated process.

Uses of Vectors and Host Cells

The recombinant host cells expressing the peptides described herein havea variety of uses. First, the cells are useful for producing a enzymeprotein or peptide that can be further purified to produce desiredamounts of enzyme protein or fragments. Thus, host cells containingexpression vectors are useful for peptide production.

Host cells are also useful for conducting cell-based assays involvingthe enzyme protein or enzyme protein fragments, such as those describedabove as well as other formats known in the art. Thus, a recombinanthost cell expressing a native enzyme protein is useful for assayingcompounds that stimulate or inhibit enzyme protein function.

Host cells are also useful for identifying enzyme protein mutants inwhich these functions are affected. If the mutants naturally occur andgive rise to a pathology, host cells containing the mutations are usefulto assay compounds that have a desired effect on the mutant enzymeprotein (for example, stimulating or inhibiting function) which may notbe indicated by their effect on the native enzyme protein.

Genetically engineered host cells can be further used to producenon-human transgenic animals. A transgenic animal is preferably amammal, for example a rodent, such as a rat or mouse, in which one ormore of the cells of the animal include a transgene. A transgene isexogenous DNA which is integrated into the genome of a cell from which atransgenic animal develops and which remains in the genome of the matureanimal in one or more cell types or tissues of the transgenic animal.These animals are useful for studying the function of a enzyme proteinand identifying and evaluating modulators of enzyme protein activity.Other examples of transgenic animals include non-human primates, sheep,dogs, cows, goats, chickens, and amphibians.

A transgenic animal can be produced by introducing nucleic acid into themale pronuclei of a fertilized oocyte, e.g., by microinjection,retroviral infection, and allowing the oocyte to develop in apseudopregnant female foster animal. Any of the enzyme proteinnucleotide sequences can be introduced as a transgene into the genome ofa non-human animal, such as a mouse.

Any of the regulatory or other sequences useful in expression vectorscan form part of the transgenic sequence. This includes intronicsequences and polyadenylation signals, if not already included. Atissue-specific regulatory sequence(s) can be operably linked to thetransgene to direct expression of the enzyme protein to particularcells.

Methods for generating transgenic animals via embryo manipulation andmicroinjection, particularly animals such as mice, have becomeconventional in the art and are described, for example, in U.S. Pat.Nos. 4,736,866 and 4,870,009, both by Leder et al., U.S. Pat. No.4,873,191 by Wagner et al. and in Hogan, B., Manipulating the MouseEmbryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1986). Similar methods are used for production of other transgenicanimals. A transgenic founder animal can be identified based upon thepresence of the transgene in its genome and/or expression of transgenicmRNA in tissues or cells of the animals. A transgenic founder animal canthen be used to breed additional animals carrying the transgene.Moreover, transgenic animals carrying a transgene can further be bred toother transgenic animals carrying other transgenes. A transgenic animalalso includes animals in which the entire animal or tissues in theanimal have been produced using the homologously recombinant host cellsdescribed herein.

In another embodiment, transgenic non-human animals can be producedwhich contain selected systems that allow for regulated expression ofthe transgene. One example of such a system is the cre/loxP recombinasesystem of bacteriophage P1. For a description of the cre/loxPrecombinase system, see, e.g., Lakso et al. PNAS 89:6232-6236 (1992).Another example of a recombinase system is the FLP recombinase system ofS. cerevisiae (O'Gorman et al. Science 251:1351-1355 (1991). If acre/loxP recombinase system is used to regulate expression of thetransgene, animals containing transgenes encoding both the Crerecombinase and a selected protein is required. Such animals can beprovided through the construction of “double” transgenic animals, e.g.,by mating two transgenic animals, one containing a transgene encoding aselected protein and the other containing a transgene encoding arecombinase.

Clones of the non-human transgenic animals described herein can also beproduced according to the methods described in Wilmut, I. et al. Nature385:810-813 (1997) and PCT International Publication Nos. WO 97/07668and WO 97/07669. In brief, a cell, e.g., a somatic cell, from thetransgenic animal can be isolated and induced to exit the growth cycleand enter Go phase. The quiescent cell can then be fused, e.g., throughthe use of electrical pulses, to an enucleated oocyte from an animal ofthe same species from which the quiescent cell is isolated. Thereconstructed oocyte is then cultured such that it develops to morula orblastocyst and then transferred to pseudopregnant female foster animal.The offspring born of this female foster animal will be a clone of theanimal from which the cell, e.g., the somatic cell, is isolated.

Transgenic animals containing recombinant cells that express thepeptides described herein are useful to conduct the assays describedherein in an in vivo context. Accordingly, the various physiologicalfactors that are present in vivo and that could effect substratebinding, enzyme protein activation, and signal transduction, may not beevident from in vitro cell-free or cell-based assays. Accordingly, it isuseful to provide non-human transgenic animals to assay in vivo enzymeprotein function, including substrate interaction, the effect ofspecific mutant enzyme proteins on enzyme protein function and substrateinteraction, and the effect of chimeric enzyme proteins. It is alsopossible to assess the effect of null mutations, that is, mutations thatsubstantially or completely eliminate one or more enzyme proteinfunctions.

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith specific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the above-described modesfor carrying out the invention which are obvious to those skilled in thefield of molecular biology or related fields are intended to be withinthe scope of the following claims.

                   #             SEQUENCE LISTING<160> NUMBER OF SEQ ID NOS: 5 <210> SEQ ID NO 1 <211> LENGTH: 2002<212> TYPE: DNA <213> ORGANISM: Human <400> SEQUENCE: 1cgcctcccag cgactctcgg cagtgccgga gtcgggtggg ttggcggcta ta#aagctggt     60agcgaagggg aggcgccgcg gactgtcctt tcgtggctca ctccctttcc tc#tgctgccg    120ctcggtcacg cttgctcttt caccatgcct ggatcacttc ctttgaatgc ag#aagcttgc    180tggccaaaag atgtgggaat tgttgccctt gagatctatt ttccttctca at#atgttgat    240caagcagagt tggaaaaata tgatggtgta gatgctggaa agtataccat tg#gcttgggc    300caggccaaga tgggcttctg cacagataga gaagatatta actctctttg ca#tgactgtg    360gttcagaatc ttatggagag aaataacctt tcctatgatt gcattgggcg gc#tggaagtt    420ggaacagaga caatcatcga caaatcaaag tctgtgaaga ctaatttgat gc#agctgttt    480gaagagtctg ggaatacaga tatagaagga atcgacacaa ctaatgcatg ct#atggaggc    540acagctgctg tcttcaatgc tgttaactgg attgagtcca gctcttggga tg#ggcttcgt    600gggacacata tgcaacatgc ctatgatttt tacaagcctg atatgctatc tg#aatatcct    660atagtagatg gaaaactctc catacagtgc tacctcagtg cattagaccg ct#gctactct    720gtctactgca aaaagatcca tgcccagtgg cagaaagagg gaaatgataa ag#attttacc    780ttgaatgatt ttggcttcat gatctttcac tcaccatatt gtaaactggt tc#agaaatct    840ctagctcgga tgttgctgaa tgacttcctt aatgaccaga atagagataa aa#atagtatc    900tatagtggcc tggaagcctt tggggatgtt aaattagaag acacctactt tg#atagagat    960gtggagaagg catttatgaa ggctagctct gaactcttca gtcagaaaac aa#aggcatct   1020ttacttgtat caaatcaaaa tggaaatatg tacacatctt cagtatatgg tt#cccttgca   1080tctgttctag cacagtactc acctcagcaa ttagcaggga agagaattgg ag#tgttttct   1140tatggttctg gtttggctgc cactctgtac tctcttaaag tcacacaaga tg#ctacaccg   1200gggtctgctc ttgataaaat aacagcaagt ttatgtgatc ttaaatcaag gc#ttgattca   1260agaactggtg tggcaccaga tgtcttcgct gaaaacatga agctcagaga gg#acacccat   1320catttggtca actatattcc ccagggttca atagattcac tctttgaagg aa#cgtggtac   1380ttagttaggg tggatgaaaa gcacagaaga acttacgctc ggcgtcccac tc#caaatgat   1440gacactttgg atgaaggagt aggacttgtg cattcaaaca tagcaactga gc#atattcca   1500agccctgcca agaaagtacc aagactccct gccacagcag cagaacctga ag#cagctgtc   1560attagtaatg gggaacatta agatactctg tgaggtgcaa gacttcaggg tg#gggtgggc   1620atggggtggg ggtatgggaa cagttggagg aatgggatat ctggggataa tt#ttaaagga   1680ttacatgtta tgtaaatttt tatgtgactg acatggagcc tggatgacta tc#gtgtactt   1740gggaaagtct ctttgctcta tttgctgaca tgcttcctgt tgtggtctgg cc#aatgccaa   1800atgtactcga atgatgttaa gggctctgta aaacttcata cctctttggc ca#tttgtatg   1860catgatgttt ggtttttaaa catggtataa tgaattgtgt acttctgtca ga#agaaagca   1920gaggtactaa tctccaatta aaaaattttt taacatgtaa aaaaaaaaaa aa#aaaaaaaa   1980 aaaaaaaaaa aaaaaaaaaa aa            #                  #               2002 <210> SEQ ID NO 2 <211> LENGTH: 478 <212> TYPE: PRT<213> ORGANISM: Human <400> SEQUENCE: 2Met Pro Gly Ser Leu Pro Leu Asn Ala Glu Al #a Cys Trp Pro Lys Asp 1               5   #                10   #                15Val Gly Ile Val Ala Leu Glu Ile Tyr Phe Pr #o Ser Gln Tyr Val Asp            20       #            25       #            30Gln Ala Glu Leu Glu Lys Tyr Asp Gly Val As #p Ala Gly Lys Tyr Thr        35           #        40           #        45Ile Gly Leu Gly Gln Ala Lys Met Gly Phe Cy #s Thr Asp Arg Glu Asp    50               #    55               #    60Ile Asn Ser Leu Cys Met Thr Val Val Gln As #n Leu Met Glu Arg Asn65                   #70                   #75                   #80Asn Leu Ser Tyr Asp Cys Ile Gly Arg Leu Gl #u Val Gly Thr Glu Thr                85   #                90   #                95Ile Ile Asp Lys Ser Lys Ser Val Lys Thr As #n Leu Met Gln Leu Phe            100       #           105       #           110Glu Glu Ser Gly Asn Thr Asp Ile Glu Gly Il #e Asp Thr Thr Asn Ala        115           #       120           #       125Cys Tyr Gly Gly Thr Ala Ala Val Phe Asn Al #a Val Asn Trp Ile Glu    130               #   135               #   140Ser Ser Ser Trp Asp Gly Leu Arg Gly Thr Hi #s Met Gln His Ala Tyr145                 1 #50                 1 #55                 1 #60Asp Phe Tyr Lys Pro Asp Met Leu Ser Glu Ty #r Pro Ile Val Asp Gly                165   #               170   #               175Lys Leu Ser Ile Gln Cys Tyr Leu Ser Ala Le #u Asp Arg Cys Tyr Ser            180       #           185       #           190Val Tyr Cys Lys Lys Ile His Ala Gln Trp Gl #n Lys Glu Gly Asn Asp        195           #       200           #       205Lys Asp Phe Thr Leu Asn Asp Phe Gly Phe Me #t Ile Phe His Ser Pro    210               #   215               #   220Tyr Cys Lys Leu Val Gln Lys Ser Leu Ala Ar #g Met Leu Leu Asn Asp225                 2 #30                 2 #35                 2 #40Phe Leu Asn Asp Gln Asn Arg Asp Lys Asn Se #r Ile Tyr Ser Gly Leu                245   #               250   #               255Glu Ala Phe Gly Asp Val Lys Leu Glu Asp Th #r Tyr Phe Asp Arg Asp            260       #           265       #           270Val Glu Lys Ala Phe Met Lys Ala Ser Ser Gl #u Leu Phe Ser Gln Lys        275           #       280           #       285Thr Lys Ala Ser Leu Leu Val Ser Asn Gln As #n Gly Asn Met Tyr Thr    290               #   295               #   300Ser Ser Val Tyr Gly Ser Leu Ala Ser Val Le #u Ala Gln Tyr Ser Pro305                 3 #10                 3 #15                 3 #20Gln Gln Leu Ala Gly Lys Arg Ile Gly Val Ph #e Ser Tyr Gly Ser Gly                325   #               330   #               335Leu Ala Ala Thr Leu Tyr Ser Leu Lys Val Th #r Gln Asp Ala Thr Pro            340       #           345       #           350Gly Ser Ala Leu Asp Lys Ile Thr Ala Ser Le #u Cys Asp Leu Lys Ser        355           #       360           #       365Arg Leu Asp Ser Arg Thr Gly Val Ala Pro As #p Val Phe Ala Glu Asn    370               #   375               #   380Met Lys Leu Arg Glu Asp Thr His His Leu Va #l Asn Tyr Ile Pro Gln385                 3 #90                 3 #95                 4 #00Gly Ser Ile Asp Ser Leu Phe Glu Gly Thr Tr #p Tyr Leu Val Arg Val                405   #               410   #               415Asp Glu Lys His Arg Arg Thr Tyr Ala Arg Ar #g Pro Thr Pro Asn Asp            420       #           425       #           430Asp Thr Leu Asp Glu Gly Val Gly Leu Val Hi #s Ser Asn Ile Ala Thr        435           #       440           #       445Glu His Ile Pro Ser Pro Ala Lys Lys Val Pr #o Arg Leu Pro Ala Thr    450               #   455               #   460Ala Ala Glu Pro Glu Ala Ala Val Ile Ser As #n Gly Glu His465                 4 #70                 4 #75 <210> SEQ ID NO 3<211> LENGTH: 28001 <212> TYPE: DNA <213> ORGANISM: Human <220> FEATURE:<221> NAME/KEY: misc_feature <222> LOCATION: (1)...(28001)<223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 3ccatttttcc cgccatcact gtctttaaat tagtccatcg gaattagttt ag#cctgtgca     60gtctaaccct agccaataag ggaacgacac agcagtgggg accacgtgcg tc#aggaataa    120gaaccccttt ccctccctcg tccaagtgtg cactcaccat tgctccatct gt#aagggtgc    180acccttctat agaagtacct tgccttgctg agaattaaaa agaaaatttt at#attcgact    240gctatttctt ttgcagcatg gaaactttat ttataacaag atcttctgta tc#taattact    300aacccttttt gttctccatt gcttggcttc ccagtaatca ataatcatgc tc#actttgct    360taattgaaga ttaacgtgat caaaaagacg gtctgttcct tgtagaaatt tc#cggttgtg    420taagatggtc attctcatga ccgtctggct aatcatttcc cattatgtac tc#ctggagtt    480ggaattattt gcgattccta acgacaaaac tgtatcttct ttcttgtgtt tg#tccttact    540gcctttcagc atattccaat atgccaagaa ttttaatctc ctaccccacc cc#aaattgct    600gttgatcata atcaggcaat gtctctctct ctgtttacta tctagttact tt#acatacat    660atgaagtgag tcatgggcaa tactgtggaa tggaaatcat tactgagtgg tc#ctcttccc    720ccaagtcatt tatgccacca cttcacagtg gttccatttc caatatattt tg#ccactttg    780ctgctgagaa tgtgtcttac taggttagca tctatagtgg ttaaaagaat ct#cccataac    840aataattgtg tgaatcacag aattaccaat gaccccttat caatagcatt cc#tgttaatt    900aaattgagat ggggagagat acaaacaact ccgaacctca ctcatggtcc cc#caccaaag    960ctaagtatta tggcttctct ctctgaccag atagaggcag agtttattgc aa#agccacaa   1020gtgtcctcct ttggattccc ccaaatagtg tttcagtgaa ttcctctagc tt#gaattgct   1080cctctctatt tgctggggga gttaggcagt ccgtatccga tggatttact at#gccgacaa   1140ttacgtggcc tttccacagc cttttacttg gcaggtacca catatgaagc tt#agaagata   1200cagtgggcaa caggccaaat ggagtccctt tcctcagagt gcatggcctg gc#aaaaatcc   1260ttgaattcag tatcaacttc ccttcacagg caaggctctg caccctcccc ac#ggatgcct   1320aatcctgaaa ccattttgtt ttaggtttag ttagaaagct ttgtctcaag ag#cacttttg   1380tttgttctgt tttctttaag tcaaggtagt tttgaataaa ggagacaatn at#ttgagtat   1440ttacaaatcg ggtatttaga ctatttacac atatacaagt tctgggtgaa gt#attctgct   1500ccaatttgca atctacgcac actttgctag aaaacgttaa gactgaattc aa#atcaagta   1560cagtatttca gaaatctttc aggtgaagcc tagttctggt tgctaggcaa cc#tgacagac   1620tcccaagctg ggaccacctc gcctcccaca tttgaccatc tctccagcgg tg#ggacgcgg   1680agtacccatt ggcccgcatc tcctctcact tagtcccaat tggtcggaga ac#ctctcact   1740ccgctcccgt tggctctcgc cgtatctcgc agctccgtca ttggcaactg gg#ctctcgtg   1800ccacctcacg tcagtctctc acaccacttc ctcggccctg agactttgtc cc#cgcctctt   1860ctccccgccc ttccagccac gagggaaaat cctagcgagt catcgcctct ag#tttccttt   1920tgattggtag aagccggact ggggggcggg cgctgccggg caactctacc gg#ccgcgatt   1980ggctgtggga gccaccgtcc cgcctcccag cgactctcgg cggtgccgga gt#cgggtggg   2040ttggcggcta taaagctggt ggcgaagggg aggcgccgcg gactgtcctt tc#gtggctca   2100ctccctttcc tctgctgccg ctcggtcacg cttggtgagt gtcccgcgct gg#ggagtaga   2160actgggctgc ggaggtgccg cgggcggggt gtgggccaga cagaggcggt gt#ccttgact   2220aggcccgaag gagctggggc tctgggtcag gacgtaggcg tggactttgc cc#gggaggat   2280ggggcaccgt gagcggggcc gggcgggggt tccctcgtga gggacctgag gc#cgaccgta   2340gcggatctga gaagatccga gaacacaggc gagtcgcgga ggggagaacg cg#agagggcg   2400ttgaggtcta ggtattctaa cgacagagga gttggaggtg ccagagaggc ag#ctgtgacc   2460gcctagaggt gagtgggggg tgtcaggagg gggagagaag acagttgggc ta#ccaaggcg   2520tttccagagc gttggttaag ggtggacgcc aaaggatggg caagatcctc tt#tagacgga   2580ggctggtagg ttcgcagggg gtgtgtcctg ctgccacata tagagttgat gg#aaagaagg   2640gaagtgggta gcattacttt tcttcctcag ctcaggtgca agaaagcgtt ca#caaccgtg   2700atttagacct ggctaagtac tggggctcag tctgtacttg cttcaaatct ca#tagatcac   2760tgcctcccgc cttcctgcct ccatattttt ttttgtctac gttttaaaaa at#aggcttcc   2820ttggtgttct gaaatcccac atctctctcc tactaatacc ttcgggacca gc#tttaggtg   2880atacagtgta atgggcaggc actcacagag tcctcccaca aataggtttt gg#attaagct   2940aaggatattt caaagcaagt atatggagtc tttgaaaacc cacgtctggc ct#tgaccagt   3000ggtagagaaa cgattattct gatccactct ggaggaggga tttggggaac aa#ataatgtg   3060aggttgtgcc tgtttgtcat gcttgtccct atggccttag ccttaaggca tc#agtagctg   3120ctttcactgc tcacctctgc tgcagctccc caccttcccg aggatgctct tg#ccacctgc   3180tgcagtagga tgatgtgttc tggttgctgc taactaacat ttgctctgtt tt#aggcatga   3240atatgaaaaa caatgacaag ataaacaaca aaattaagac aaatggaagt gc#tcctagag   3300ttaacagatt tttccttctg agatgtgttt tggactttat tgcacagata ct#attagatg   3360agaggcagtt gaaagtcgtt aacattaccc gtgtcagtag ttctttgcac tt#gagacacc   3420taagcagctt gtgttcttta aactttattt taaaattgca gttatttttg tg#tgaagaag   3480ggggcaggga tagcatacct tatgggaaga gagaaaggct ttctttgtgt ct#acctttgt   3540agatatttct cacctaagtt tgtaagtttg ccctttattc ggttctactt ta#gttcagct   3600caattctagt ataatcatca gtaaccccag cactcagaag gtctgactta cg#ctgtgggg   3660agggagtgta aaaggatatt ttatgtttgg agccataggc cacatcattt gg#gccttgtt   3720ttaattttgt ttttcatctt aaatatccct ccagattgct tttacatctt gt#ttctttta   3780actgtggatt gattttgaga ttttgactta gattttagat agcttttctc ag#aagaaata   3840aacgcaaaaa cccgatattg ttgtaacatc agtttcctgt gtcctctaga at#catttaaa   3900acctggttgg atcttccata atccagtgga attggatatg agatgtagct gg#agaagttt   3960gttttgctac atatcagaat ctccaattag tttcatttag aaaggaatat ag#ccttataa   4020ttttatgctg ggttactgtg gaaccaaata tcatagaagg atgtgtgata tt#tttatgtt   4080tttcaagaag gtagtataga tttaaaaggt gggatacata ttacctgtcc ta#atgatagg   4140actagatttt tttttttttt ttttttgggg agacagaatc tcgctctgtc gc#ccaagctg   4200gagtgcagca gcgtgatctc ggctcactgc aacttatgcc tcccagtgat tc#tcctgcct   4260cagcctccca agtagctggg actaccggca tgtgccacca cacccagcta at#ttttttgt   4320atttttagaa gagatggggt gtcaccatgt tggtcagact ggtcttgaac tc#ctgacctc   4380aaatgatccg tccgccttgg cctcccaaag tgctgagatt acaggcgtga gc#caccatgc   4440ctggctagaa ctagactctt aatctcttca tcctaatgca tggcgtgtgt tg#atgttcac   4500ttaatgtctg tcaactgggt gtagttacac cagtagcgga gaggctaatc tt#tgaaagcc   4560tgaagtgttg tcttcatctt tgcagggttt ttagttgtgg gtgcatatgg ga#atgattgt   4620aagaccaaca aatgttttct gattccatat gggcttctta catttttcac ct#tggaatct   4680gggaacaatt gaaacctacc atatgccttg aacagtagca gtaaagagcc ag#tttcttta   4740aactagacat tatggtgctg cagctcatct caaaactgat agcaggctac tc#tggacaca   4800ctacatatag agtagccctg ctctgcaagg agcagtaata aattaaaaaa aa#aattaaaa   4860agtgatagca gaaagcactt actactgagg gctgctacaa gtattaaatc ta#aaagattt   4920gtcctctagt agttataact ccaaattcag ccactgaaaa atgtgacatt tg#agtaccct   4980ttacttcaag gtctcaaagg gatttcaaaa aatcaaaata tatagcccct ct#cccaaaag   5040aagtgtagga atcctgtatg gataagaaga ctgcccataa ctagttttcc at#agagagta   5100ggctatgtag acttgggtat gaatgaccta cctctgtaga agtgcaggtc cc#tgattaga   5160aaacttattt tctgtgtgat ttatcgagga aagcttccag gaagaggtga ct#tagaacag   5220ggccttgaag atgagtagaa tctctgatac gcagaccagt aactctggga gg#aggcaggg   5280atgtccatgc tttttacttg gagaactata ccagagtgta caggtttgag ca#agtctttc   5340ttaacattag tttttacttg cttgctccta aggaggaaag gttgccaact tg#ttcttaat   5400ttcctagatt tatctcctgt aacaatgaga aagatcaata ggtaactgtt ta#tattttat   5460agtttacata ccaaaatgtg taggcaatga acttctccaa ccacttcttt ga#atcaaggc   5520taaggaggga gccagaagga agtattcaga acactgagta aactccagaa ga#aactacca   5580ttgcataaat ctggttggcc ctaggcagtc ttatcattct tgtgttttag tc#tttgccag   5640actcaaagtg cctatatttc atcccatgag tctgcaaacc tgctttgtgg ta#acctgcct   5700ggctacttgc cattcattaa ctgcttcttg acccatgttg attccctctg tc#acttactc   5760tgaaaagacc tgttagaaat aagcttgtga tctgcttgag actttggcaa ta#ctggttta   5820gccagaatag agaaatcctt aagtagcaca gcaatccttt ctgaatcttc ta#tttgtttc   5880ttctttgttc tctgtgtctc tcccacctaa catccctctc caatttaagt aa#tcaaaata   5940gaaagagggg cccaggcaag gtggcccacg cctataatcc cagcactttg gg#aggccaaa   6000gtgggtggat tggtttagcc caggagttgg agaacagcct gggaaagatg gc#aaaacccc   6060atctctacaa aaaatacaaa aatcagctgt gtattgtggc atgtgcctgt ag#tcccagct   6120acttgcgggg tctgagacag gaggatcact tgagcctggg aggtcgaggt ta#cagtgagc   6180agtgactgga atgctactgc attccagtct gggtgacaga gggagaccct gt#ctcaaaaa   6240aaaaaaaaaa tttgagggaa tataggcagt gcaaggaaag gcagaatata gg#cagttcaa   6300ggaaaatttc cttgatacaa gtagtgtcaa atgcatatac atacatgaac at#caagaaga   6360aatattatta tttaagtagt cttaacatgg agaaggaatc ttgtttttca ag#aactggtc   6420tctgtggtct gcttaatttg cagaagacaa aggcataatt tgagataata aa#gaacaaag   6480ataggttatt ttctcaaagt atgtataatt acagttaatt agagacattt tt#ggaatatt   6540gtagtattct ttgcctacaa aactcaagat ctatttcttt ttatggggca gg#ggggcgta   6600ggtgggtagt aaacttagtt aatgaagtaa aaggcgctac gactgaagag ct#cttaaatt   6660atgtaattat gtaaaaaaag taaagcttta ttaaatatta ataacatccg aa#tgtagtta   6720ccagtgaatc cattaagggc agatgctaaa tttgccagta attaaataga ga#gcagagga   6780aatggtgtat gctgtgttaa acatagaagt tgccatctca agtaacaatc ag#tctttcaa   6840aacagatgga ctgaagaata tgttccagtc accttcgcaa attatttcta ct#taatttac   6900ataataatgt ttaatgctcc tttgtctaaa tgcttaattt tttaacataa gc#agtaagag   6960ggaaaatcac tttataaaag gttgggaggg tgaaggtggc agtgttgaaa at#gattaggt   7020cttgctagaa aaaatacctt tattttcttt gaaaaacact tataagaact at#aagaacta   7080aggtaatagt cagtgtattg gtgctttgtg ttacaaagtg tcttcacata tt#ttatcatc   7140tcagcaatcc ttcacaatga tctggggagg gcaactgtat tagcttcatt tt#atagatga   7200ggaaactgag gtccagaatt gctgccaaag ccacaatctg ttacatgcag tg#caggctct   7260tgactgcata tatctcttta ctctagaaat ttgctaactc attacaactt gt#ttatattc   7320ctttccccca attcttgaaa accttggttt aaagcctcaa ttggtgacat gg#gcttctta   7380tttccttgag gtttttttgt ttattccttc ctgcaatagt aggcttctta ta#tccgttta   7440ttaccaggac tgaacctttc actataaggg ctatgaaaat aagggggaaa at#gttctata   7500agctttaagt atgatttttt ctaagcaaat gtcaaattct attctgcata at#gtaattgg   7560ataaggaatt gcttatttta actcactttg aattggattc attagtattt ga#atttgggt   7620aggatttata actttaaaag cannnnnnnn nnnnnnnnnn nnnnnnnnnn nn#nnnnnnnn   7680nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn#nnnnnnnn   7740nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn#nnnnnnnn   7800nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn#nnnnnnnn   7860nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn#nnnnnnnn   7920nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn#nnnnnnnn   7980nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn#nnnnnnnn   8040nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn#nnnnnnnn   8100nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn#nnnnnnnn   8160nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn#nnnnnnnn   8220nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn#nnnnnnnn   8280nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn#nnnnnnnn   8340nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn#nnnnnnnn   8400nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn#nnnnnnnn   8460nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn#nnnnnnnn   8520nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn#nnnnnnnn   8580nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn#nnnnnnnn   8640nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn#nnnnnnnn   8700nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn#nnnnnnnn   8760nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn#nnnnnnnn   8820nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn#nnnnnnnn   8880nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn#nnnnnnnn   8940nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn#nnnnnnnn   9000nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn#nnnnnnnn   9060nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn#nnnnnnnn   9120nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn#nnnnnnnn   9180nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn#nnnnnnnn   9240nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn#nnnnnnnn   9300nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn#nnnnnnnn   9360nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn#nnnnnnnn   9420nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn#nnnnnnnn   9480nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn#nnnnnnnn   9540nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn#nnnnnnnn   9600nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn#nnnnnnnn   9660nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn#nnnnnnnn   9720nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn#nnnnnnnn   9780nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn#nnnnnnnn   9840nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn#nnnnnnnn   9900nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn#nnnnnnnn   9960nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn#nnnnnnnn  10020nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn#nnnnnnnn  10080nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn#nnnnnnnn  10140nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn#nnnnnnnn  10200nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn#nnnnnnnn  10260nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn#nnnnnnnn  10320nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn#nnnnnnnn  10380nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn#nnnnnnnn  10440nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn#nnnnnnnn  10500nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn#nnnnnnnn  10560nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn#nnnnnnnn  10620nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn#nnnnnnnn  10680nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn#nnnnnnnn  10740nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn#nnnnnnnn  10800nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn#nnnnnnnn  10860nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn#nnnnnnnn  10920nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn#nnnnnnnn  10980nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn#nnnnnnnn  11040nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn#nnnnnnnn  11100nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn#nnnnnnnn  11160nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn#nnnnnnnn  11220nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn#nnnnnnnn  11280nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn#nnnnnnnn  11340nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn#nnnnnnnn  11400nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn#nnnnnnnn  11460nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn#nnnnnnnn  11520nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn#nnnnnnnn  11580nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn#nnnnnnnn  11640nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn#nnnnnnnn  11700nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn#nnnnnnnn  11760nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn#nnnnnnnn  11820nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn#nnnnnnnn  11880nnnnnnnnna ctttatcaaa aaattgatgg ggagagtttg ttgaagctca ga#gtgaggat  11940ggatgtagaa catttcaagt gcttcatatc cagaaaatca gtagtcctcc at#ctgagttg  12000tagacacagg aaaggagttg aagatgaatg gagtaggaat gtaaaagcct ta#tctttacc  12060ctcctcagct ttaggtctta acaagaatga gcctccctta gtctttcttt at#gcccctgt  12120ccctgaatgt tggtgatgac attgtttttc ctgtattgaa tacaaaaata tg#gccagtaa  12180tttaggaatc aagaggatat aattcggaag tagactgttg tgtttaggag tt#tttctttc  12240cattgtggaa ttgagtagca gcggtatata tgctatgtct ggtaaaatgg gc#catacagt  12300agtctaagac atgaggagac cttaaggagc ttggacttag ttgaggtgac ca#gactattt  12360aatctgctta ggtgccacag caaaatacca tagagtaggt ggtttaaaca gc#agacattt  12420atgatctcat aggtttgcag tctggaagtc agggtgccag cgtggttggt tc#ccgatcag  12480ggctctcctc ctggattgcc cgtgtcctca catggcatag agagagtatg ac#agcatgag  12540caagctctcg ttttatcttc ttataagagc actgatccca tcatgagggc cc#cattctca  12600tgacctcatc taaacctgat tattttccaa aggccccatc tccaaatgcc at#cacattga  12660gagttaaggc ttcaacatat gaatttggtg gggaaaccca gacatttcaa tc#cataattc  12720aggcagatat ttgggaagta acacagttga agcactgaat gctatatttc gt#actatcta  12780aagaatctag gatgtaataa atttaagatg cttcattgcc aattaaatta ag#atacaatg  12840cttttttgat tacttagaat tttttaaaga gctcttttag agttagacat ag#atttttgt  12900catatgtcac ttgcacattc aataagatgg aaaacacaag tgaaaaaaca ca#taaggaat  12960tgctaaattt cacatattta gagtctgcct tctgaattgt ttttggagtc ag#agttgtta  13020atacctgtaa ttttccgtta aacatcctct gtgccgccaa gagaattggt ga#tgtagcat  13080tcctttcaag atcccaaaaa agaatgcgaa ggttttggtg ctggccttca gc#tttgcaat  13140tatgcaaagc cagcctactt tgactgctgc ttagggattc cccatcttct ac#ttccttcc  13200cagtccattt ggttcctaga gggtgaaatg aatgctccag tatcatttct gg#gaatttct  13260ttcaggctgt tgactgtcat atgcaaatgt catgctggca gttttgttat tt#tcccatgt  13320gtaagcaatg acaacatcat aattggcttc tgtctgatag caattgtaag ag#gaatccca  13380atttctgaaa tgttacccaa aaaagtgact ttaattgacg aagtatgatg at#gtagaagg  13440ataggcaaga aatgcaaaag gtaatttaga aaggtttcat gggtaaaatg tg#acctatgt  13500gatctagggc tataaaggat ttcaataagc agaagcacga ggtgggttgt tg#aagaaagc  13560actaaatgtt tttggataaa gaatataata atttgagagt aaagggtaga gg#gagggtta  13620tgtaggtaag tagttgtaag atggggaaag attgggtagt atttagcatt ta#tccttaat  13680gttgacttca gtgtagttct ctttgtgtgt tttctagtat aaactgcata ca#tgaaagtt  13740aagaatcttg tgttaagtcc catataggaa ggaagtagat aggaaaacca aa#ctggaaaa  13800atgtatggag atgttggtga aatgacagga acgaaagcag cttgtctgag ct#tgatctct  13860tcacttcctc agtggtggtt ctgagcgctg gtttggctga actccactta cc#agggaaaa  13920gggcataaag taaacagggt ttgtgtggaa gaagtggagt agaacaaagt gg#agaggatc  13980tctgttcatt tagtgtatct gacagtgtgc ttgtcaagtc ataaaacact tg#aggatgga  14040aatctggaag tcattgtata cattttcttc tttccctaac atctagtcag tt#acagtttc  14100tgccagttct tttgcttttt ccatgttttt ggaggctgtt cctcttcgct cc#acatgtag  14160taaatgctct agttcatgac ccatgtctta tctggactgc catgtcagct tc#ctaactca  14220tccattcaca gcaccagtga ctgtaaaaca gcattagtga ggataaaaca gt#ggctgtca  14280aacttttttg actgtggccc ccagtaaaaa tacactttgt attgcaactt at#gtatactt  14340tatatatgta tgaataatta aaacaaaagg ttgattcaag aaaaatcttt ac#atttaccc  14400tgtgccatgc aatcttatat cttgtattct tttctgtttc atttttttaa at#gtgtgctt  14460gccatccact aaattgattc cggagttgga aaaacactga cctgacaact aa#tatcacca  14520tgttattcct taaactctcc gatggcttct tactatcttc atgataaatt tg#aagccctc  14580aacatcagca taccagaacc ttcatgacct aacccttacc tagttattct aa#tctattat  14640ttacctgatc cactcagctc acatttcatt ccaatagaca agtaaagttt tt#tgtaattc  14700cttgtagctt gcctttcttc atggtgtcca ctctgttgaa aatctactac cc#tccatttc  14760ttcagtgctt tactgcttac tcctacccat tcctggggct caagtcaggc cc#ctataacc  14820aggatgcttt tcctaacact ccttgcccta ccaccaggct gggttaggta gt#tctccatt  14880atataatgtg gttctcaatg ttgttacctg tttattatta tgtgtttttc tc#ttattgtc  14940ccataaaata gtgaatattc gagaggataa ggaagtctcc cattaagcat cc#ctaatgtt  15000tagtatgtaa catgttggca ttggttggat gaatgagaaa aaaaaaagat tc#ttctgttt  15060ggaaggaaga tacaactggt atcccttaag tcttttcttt tttttttttt tt#ttcctttc  15120tctatagaca aggtctcacc atcacccagg ctggagtgca gtggtgcaat ca#cagctcac  15180tacacccttg tactcctggg ctcaagtgat cctgctacct cagcctccct ag#tagctggg  15240actgcaggca tgcaccacca tgctcagctc attttaaaaa aatttttttt gt#tgagacag  15300agtcttgcta tgttgcctag gctggtcttg aactcctggg ctcaagtgat cc#tcctgcct  15360cagcctccca gagtgctagg attataggca tgatccactg cacctggccc ct#taagacct  15420ttaattgcag agcagcagag gacaaatgac ataaatacag gatttgactt tc#atttttaa  15480gtatcaaatt agtgatgggt tgacaaacaa gtcatacaga atgttcatga at#cagttcgg  15540cccaggtaac tcataaccca agacctttgg gtcaatgaaa ttctgccacc ta#agtagcac  15600catccaatga tgtcatacct aaaaaggaaa ttgagttgta gaattttagg tt#ttaggatt  15660ctttctctaa aactgaggag ctgtgccact cttcaaagcc tcacaattac at#ttcattgg  15720ttcttatgcc atctgggttc tggttagagg gctgatggaa gtactcaaga aa#tattggaa  15780gtactcaaga aatattagaa ggtgggaaga aggtacctct cttgttcttg tc#agtggcag  15840caccaacagt gggactttgg gtctctgggt tccagctcag cagcagaggt ac#tagtactg  15900tagctccagc agcttcagca ggagtgcagg ctcatgggat cagagaacca cc#ttttccgc  15960tttgttcttc cagcccagcc aacaagtttg tagctatttc cctgcattaa aa#ctcccctc  16020tgtttgaaat atctatagta atttttcttt tcctgactaa tacaacctgt ta#aagaagct  16080gaagctctgg taagttaaat gcccaacaat ggtcttgagt agctagtgat tt#ttgttgct  16140attggtaagt aaatctagac actacttttt agtccctttt ttaaaagagg ac#tggtttat  16200ctatgatgaa tacatgattg attgattgat tgattgattg atttttactt tt#tctttttt  16260tttttttgag acggagtctt gctctgtcac ccaggctgga gtgcagtaac at#gatctctg  16320ctcactgcaa gctcctcctc ccgggttcac gccattctcc tgcctcagcc tc#ctgagtag  16380ctggggctac aggcatctgc caccacgccc ggctaatttt tttgtatttt tt#gtagagac  16440ggggtttcac catgttagcc aggatggtct cgatctcctg accttgtgat cc#gcctgcct  16500cagcctccca aagtgctgag attacaggca tgagccacca cgcccggcct aa#tttattaa  16560aactttcggg tggtcaggta attctgattt gtcagccata tttctaaatt at#caatnnnn  16620nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn#nnnnnnnn  16680nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn#nnnnnnnn  16740nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn#nnnnnnnn  16800nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn#nnnnnnnn  16860nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn#nnnnnnnn  16920nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn#nnnnnnnn  16980nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn#nnnnnnnn  17040nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn#nnnnnnnn  17100nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn#nnnnnnnn  17160nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn#nnnnnnnn  17220nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn#nnnnnnnn  17280nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn#nnnnnnnn  17340nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn#nnnnnnnn  17400nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn#nnnnnnnn  17460nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn#nnnnnnnn  17520nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn#nnnnnnnn  17580nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn#nnnnnnnn  17640nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn#nnnnnnnn  17700nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn#nnnnnnnn  17760nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn#nnnnnnnn  17820nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn#nnnnnnnn  17880nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn#nnnnnnnn  17940nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn#nnnnnnnn  18000nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn#nnnnnnnn  18060nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn#nacaggca  18120cacaccacca tgcctggcta attttttgta tttttagtaa cagggtttca cc#atgttagc  18180caggctggca tcgaattcct gacctcaggt gatccgcccc cctcaacctc cc#aaagtgct  18240gggattacag gcgtaagcca ccatgcctgg cctgtattta atcttcatag ca#gttttatg  18300aggtaggtgg tgtcatcccc actttacaga gaagtgggtt aatgtagggt tc#aaatgata  18360aatagtaact tgctgatagt cactggcaat tttaatttgt cttcagtgta gt#agagtaac  18420tgtgaactgt tagagttatg aaactgacat ggaaagttgt ataccaaagg ag#tcttagga  18480ctgtccatgg atactgttat gtatcatttc acttatattg gcttcagctt gc#gatttctc  18540tactgtaagt ggtgagaatt gatcagatag ttaaggaagg tccttagata at#gcagtata  18600cttattaaca tacagacatc aagaagcaga aatatataga catcttcctt tt#tggttcta  18660atagggcttc gtgggacaca tatgcaacat gcctatgatt tttacaagcc tg#atatgcta  18720tctgaatatc ctatagtaga tggaaaactc tccatacagt gctacctcag tg#cattagac  18780cgctgctatt ctgtctactg caaaaagatc catgcccagt ggcagaaagg ta#agttttac  18840ccattttcct tggttttggt atgagttgag agcagtctaa tgtactaggt at#ctttggta  18900ggcaactact ttgtgggcat tcttcattta atatcctttt accattaatt cc#tcattcac  18960caaacaacat tttcccatag tttctgggaa agtgtaattt actagaagag gt#aaactttg  19020gaactgaggt gtatctctgc aaaaatattt aggtcggttt accccttgta ag#aaaatcaa  19080agtggagaaa agaaggtaag ttgaattttg ttcatctttt gagagaggta tt#ttaacaag  19140gttttggact acagctgtga ttcagggaaa gctaatgaaa atgaattact aa#agtgatct  19200taccccaaaa ataatctttt tgcacttgac ctgtgaattt gtatttgttt tt#ttactgtt  19260atcattaatc tggaaatttg ttgaggcact gaaaggacag tatttgagtt aa#tgctatca  19320taacacatta ttacataaag tatacttttt ctgtagtcca actttgcttt tt#agaggtta  19380tgagaagggg ttaaaaatca tattcaatga caaatatcag tgaatttagt cg#ctctggat  19440aagaagcatt cttgcagtat atattaacag aatagtggtt ttctaacttt tt#tattagga  19500cccacagtaa gaagtacatg ttacattgta tgtgtatgcc agactgaaac aa#aaatgtca  19560tgacattact tacccttgct gcaagttatt cagtttgcta tttttctact gc#attttgtt  19620ttttaaaata ctcttttatt taaaaaaaat actaatcctg acccactaaa tt#gattatgt  19680aacctgctaa tgtgtatgaa tcttaaattt gaaaattagt gacatagtac at#attgtttc  19740atctttgagt gtctttttaa atgtatactt taaggtatag agaggtttca tt#atacagtg  19800tatttgtggt tgctgtttaa acatatacaa atatcctagc tttattctaa ag#tcaaactt  19860taaaatttca tggcttatat gaatttcata gtttccttgg acttctcttt ca#gagggaaa  19920tgataaagat tttaccttga atgattttgg cttcatgatc tttcactcac ca#tattgtaa  19980actggttcag aaatctctag ctcggatgtt gctgaatgac ttccttaatg ac#cagaatag  20040agataaaaat agtatctata gtggcctgga agcctttggg taagaggagc ta#ttatgagt  20100tttttccttc tatattagag catttttaat atctgttaag ctgttatttg ta#cagacctg  20160agaaattgag agtcagaaga atcttagaag tcatccagtc taatctgtgt gt#ctcagtca  20220gtgaagaatc taagtccaga gaggtggtag ttaacatgca caaattcttt ag#acatttct  20280attcagattt tctgatttat ttctttcagc tccattcatg ttgtcacgat aa#agtaactg  20340cacaagggcc tatattcact acagcagcct cttaactcct tacctctctc ag#cacccctg  20400cccccatgcc cttttccatc ctgcacactg ccacagctaa agtcagcttt tg#tactccac  20460ctgtcttttt ctcactttag gctccctagc atgctatgtg tgttcaactc gt#tctgtttc  20520tccctgtgtc tcttgtgtgt cctttctcta tctgataaaa ttatacttga ct#tttaaaac  20580ttggctcctg taataccatg acttttctaa ctaaataaac attattatgg ac#ttgaaata  20640gtattctatt cagttgatga atattcagtt gattgaatat tctattcatt ga#agccaata  20700taagtgaata taaatataaa gctacagtgc gtcttttaac ctattcaaat ca#agcaggct  20760taacttgatt atgaaaactt ttgagaaaaa gaaccatata tatacaactg tt#atgatttc  20820tatagcaatt agattgctgc tacttggctt ttaataaatg agaaaacaat ta#tatacact  20880taaagatttg aatcctaatt aggcctgctg tttagtgtaa taaaaacata gg#ctttaaac  20940actgtaaaac tgtaaaataa atctttcagg gatgttaaat tagaagacac ct#actttgat  21000agagatgtgg agaaggcatt tatgaaggct agctctgaac tcttcagtca ga#aaacaaag  21060gcatctttac ttgtatcaaa tcaaaatgga aatatgtaca catcttcagt at#atggttcc  21120cttgcatctg ttctagcaca gtaagtataa atttcaccta ctacttaact cc#ccttattt  21180gggagatgtt agatttctaa gaccaaatct agtgtcaagc atgttggtgg ta#gatcacag  21240aaaattttat cttgaggctc tctaatctgc tattgtccat tgacttgaaa ga#tgtatggg  21300ttgaggctac agttcttcca gaagtatttg ttaatttcat actggctttc ct#ggcttctg  21360ttttcatggt tttttaattc ttgacctaca gttgaaccat aaatacctgg tt#gatgaagt  21420aacttgtttt gtggcatgac tttcacaagc tctgtcattc cccacaagat ga#aaactcac  21480atgctgcaat attaaaacta agttatattc cctactgcaa tattaacact tt#gagttaga  21540tccttaaaac tttaagttag attctacttt tacttatagc ctaaattttt at#tgctactt  21600ttatagcttc ccacacgctg tagctttgga tcagttaaac ttctgaacta tt#gttacacc  21660ctacataggt actcacctca gcaattagca gggaagagaa ttggagtgtt tt#cttatggt  21720tctggtttgg ctgccactct gtactctctt aaagtcacac aagatgctac ac#cgggtaag  21780tgctgaatct ttcaacaaga atgtattgag aactgagtcc aggcacagtg gc#tcacaccc  21840gtaatcccag cagtttggga ggccgaggcg ggcagatcac ctgaggtcag ga#gttcgaga  21900ccagtctggc taacatggct gaaaccccat ctctactaaa aatacaaaaa tt#agccaggt  21960gaggtggtgc atgcctgtag tcctagctac ttgggaggct gaagtaggag aa#tcacttga  22020atccaggaga gggaggttgt ggtgagccaa gatcacacca ctgtgctcca gc#ctgggtga  22080cagagcgaga ctctgtcaaa aaaaaaaaaa aaaaatgtat tgagaactac tc#tggggaag  22140ttgatttagc agtcttctca agtgagcacc tgaatctgtc ccacagatca tt#acaatatt  22200ttagtcttca ttacttcttt cagtaggttt ttactctctg ccctaaaaat ct#atccaaaa  22260aaaaaaaaaa attctacctt atctggataa aggataggac taagttatct aa#tttttata  22320ggcttatggt cttggctata tttaaggtca cttttgtgct ttccctgagc ag#gaaagagc  22380aaaaatgtag agataaactg atgaaaactt gacattactt tttaaaatta ta#ccatgggc  22440caggtgcaat ggctcacacc tataatccca acacttcagg aggctgaggt gg#gaggattg  22500cttgaggcca gatgttcaag gccaacctga gcaacatagt gagaccccat ct#ctataaaa  22560aataataaaa ataaaataat tataccatgg attaattgta gacaagttat tt#atagtttc  22620aaattatgcc tgtttcctaa cttgtctagt ggcagatact caataataga tt#tctagtct  22680gacatcatag gagatttgtc aaataggtat catcttatct tttaactaat ca#gtagccag  22740tagttttaat gaaaatgaaa agttgttttg cctcatttgg caacatttta ct#taggcttc  22800ttttggacat gatttttcaa aaaaatcttt taatgttgaa ttattcacta tt#ttagggtc  22860tgctcttgat aaaataacag caagtttatg tgatcttaaa tcaaggcttg at#tcaagaac  22920tggtgtggca ccagatgtct tcgctgaaaa catgaagctc agagaggaca cc#catcattt  22980gggtaaaaat attaaatgtt ctttaagtta acccatttgg agggctgata tc#attaagga  23040tgctacatat acgataagga tatcaagact ttactcagta ctaatctgat gt#cagtgaaa  23100attattggga tatatgaaac ttatctttag ctttattacc agatgaattg ta#tatcataa  23160ctaattgtag atattctctc cctttccttt agtcaactat attccccagg gt#tcaataga  23220ttcactcttt gaaggaacgt ggtacttagt tagggtggat gaaaagcaca ga#agaactta  23280cgctcggcgt cccactccaa atgatgacac tttggatgaa ggagtaggac tt#gtgcattc  23340aaacatagca actgaggtaa ataaaagagt tcccatctcc atatcttagg gt#ttaggaga  23400cctaactggg atttagcaac ataaataaat gtcagtaaag aagagtaagg gc#tctgggag  23460tagattctag ctgtactatt tccaattgta taaagtgctt tgcatttgaa tt#attaatat  23520tttaagaata tacagtaaag gccgggtgcg gtggctcacg cctgtaatcc ca#gcactttg  23580ggagactgag gcaggcagat cacgaggtca ggagatcaag accatcctgt cc#aacatggt  23640gaaaccctgt ctctactaaa aatacaaaaa ttagttgggc ttggtggcac gt#gcctgtaa  23700ttccagctac tcaggaggct gagtcaggag aatggcttga accagggagt ca#gaggttgc  23760agtaagctga gatcacacca ctgcactcca gcctggcgac agagcaagat tc#catctcaa  23820aaaaaaaaaa aaaaaaaaaa aagaatatac agtaaatact aggttttatt aa#tgatacca  23880ggatttaaag gaagactgat atagagagaa ggttcatttg tggtgtgtgt ct#ttgtgaga  23940gatggagtag agggacaagg atcctttcac atctcatccc agatcatggt ca#aaatctgt  24000cctcaaattg tcaagaagta acaatcatag ctatgatttg aattcctgtt ac#ctgctagg  24060cactttactt acgttttctt atttaatcct tacaacaacc tccttgaagt tt#ataaatga  24120tactgtcctc cctttagaga tgagcctcca agaagttaca ttacttgccc ag#gattatag  24180gtagtaagta ttaaagccag gttataaact aaggacttta taaccttgaa ac#tacttatt  24240tatctgctta ctacaagttt ggtaaatgga tagtcttgct ttttgctatt at#acaaatta  24300ggtagcaagt caaaccgcca ctgtttgagt tgcaaataca agacgtaaca ag#taaaatac  24360tgttacgtgg tgggtctctg tggcaggctt cctctccccc ccatatggat aa#ttgtatac  24420taaattcacc ataaggtgaa aaatggatat tgagttccct tcatgaaaag tt#atataaaa  24480tatatattta gcataaactt ctccagagtt gtcctttatt aagtttcttt ac#agaaactt  24540taattggtgc catgattctt gtgggggaaa gaatcataag agccatcaac tt#ttttcctt  24600tcattttagc atattccaag ccctgccaag aaagtaccaa gactccctgc ca#cagcagca  24660gaacctgaag cagctgtcat tagtaatggg gaacattaag atactctgtg ag#gtgcaaga  24720cttcagggtg gggtgggcat ggggtggggg tatgggaaca gttggaggaa tg#ggatatct  24780ggggataatt ttaaaggatt acatgttatg taaattttta tgtgactgac at#ggagcctg  24840gatgactatc gtgtacttgg gaaagtctct ttgctctatt tgctgacatg ct#tcctgttg  24900tggtctggcc aatgccaaat gtactcgaat gatgttaagg gctctgtaaa ac#ttcatacc  24960tctttggcca tttgtatgca tgatgtttgg tttttaaaca tggtataatg aa#ttgtgtac  25020ttctgtcaga agaaagcaga ggtactaatc tccaattaaa aaatttttta ac#atgtaaga  25080attttgtact ttgaacaaca agattacaga aagtacctgt ggtttttgga aa#acatttct  25140agcttgggga atgtgacaac attccccagt gtggtaaaat tggggtaaaa tg#tggtaaaa  25200tgtgatacgc acaaaccctt tgaaaatagc aaaacaaaca tgcccttttt ct#aaaattga  25260taaatcctaa agaggaagaa aagagctggg acaataaaac actggctctg ga#atctggaa  25320tgttaagtcc aggccagcag tgacaaaagt tattgtaatg acctctgaac ag#agaaacac  25380tgccattgaa gaggcttctg gtatagaaaa catggtacat tcaggagctg tg#aatatagc  25440tctaggtgtg ctcctgaatc agttcatggt agattatgct gaacaacagt ga#gatgttat  25500tggaggtgtg gatgagggag tttgttgttg cagtccttct ttgcacctta tt#ttaaagaa  25560taaatgaaac atttttctgg ttactttttt aaaaatttaa aatggaaggg aa#gaataggg  25620gcagggcatt attaggctat ttctgatgct tcagtgttat aaattcaaca ta#gaggctga  25680caacctaaat tcatggtgta acacagctct tttccttttc cttttttttt tt#tttttggt  25740atctgttcaa tgaaaataag gtatgaccca agtttttacc tagtctgact ag#aagtattc  25800cacttcaagg tctgaagtag gacttttacc ttaaaaaaca acaacaaaca aa#actatcac  25860acaggataga taagaagatt ggttaaacag ttttgtgtag atctttttgg tg#ctgaacta  25920tgacatgagc cttatagatt gtaaaatagg gatagttgga actaatgtac ag#aactaaat  25980tttttaaact ttatttgctg ttaaattctg tgaagtttca gttatctaaa at#aaatatac  26040acaaatatga aatataatgt ttcagattgc aaggtaatat gtaatagtag tg#tttgtaag  26100atactcttgt ctaatattaa ctagtagtat tttgatttgt acagtcataa tt#tgttaaaa  26160tgacttcatt taacattcac tgatgtagat taataatgta agttctgatt ta#aagaatgg  26220tggcaaaatg gtgcatgtaa tacttttgca agtgttgggg agatcggtat gt#tttgaaaa  26280gagtaattta acttttgggt gccaggaaat gggttttctc aaagtccatt gc#cggcaatg  26340ggcaggcctg caaatactgg cacagagcat taatcataca ccttattaac gg#tgaggtga  26400ataactttga aataaagttt tagagaaatg tttcagatac ttgagtattc tt#tttcactc  26460ttgaactaac aacttcggca agaaatcagc taatattcta tttttaaata tg#ggcattaa  26520tttcatttca gttcgttcac tcattccatt catttatcat ttcacaaaca tt#tgaaatcc  26580taatataagc aaggtgctct gtttaaggca gaaatttgaa aatgtacaag at#atatggtc  26640ttgtctttaa ggagctgttc atctagaatg gaggaattta cactgataat ta#ttcctaca  26700cttgaaacaa agaaattaac tctcaaattg cgtggcaagc atatatagac tt#tgctataa  26760atatttatga aatgagttac tgttttcctt aaaaaagcta agactaaggg ct#ggcaatca  26820aataagagca aatttagtgg tgaacgtaga actgcccact accagctaga gt#ctccaacc  26880taaaagtccc atgttgctag tgatccccag gggttttata gaaggaatcc ct#gcattggc  26940agtaattttg gattagatga tccctaagag caccatcaag tcttaggatt ct#atgaatta  27000ggaaataaac caaattatat attttctaat actgatcagc tcatatttta tc#atcatgtc  27060atgtctggct ttcatactgg gaatacagat atagaaggaa tcgacacaac ta#atgcatgc  27120tatggaggca cagctgctgt cttcaatgct gttaactgga ttgagtccag ct#cttgggat  27180ggtatgttac atgcctattc cccgccgtcc cccaaaattt ttttctaagg tt#caatagac  27240ccaaatgaca ctttaattaa tgcaatacgc aaacttttgt aatttatcct tg#tttggata  27300tattaagaaa gatattttac ctgtctgtca ttatccgaat tgtgaattgg tt#atcttatc  27360ttgtaggaca aatggtctat tcaaaattta gtcagatgga tgacagagcc tt#ggcagatg  27420aattttaaaa aaaaattaga gcattttctt tctttatcaa agaagggaaa ag#catattct  27480ggggaaaata taacagactt cagtttccat gtttggttat agtgttgaat tc#cttcttgt  27540gaaataacaa aaaatatttt tcaggacggt atgccctggt agttgcagga ga#tattgctg  27600tatatgccac aggaaatgct agacctacag gtggagttgg agcagtagct ct#gctaattg  27660ggccaaatgc tcctttaatt tttgaacgag gtaagtgctt gggaaagcat tt#ttgttttt  27720tttagcacaa tatgctgaga aatttgaaaa tagaagtagg agctgtcgct ta#cttaatgg  27780tcattaaatg caggtactac ttgctaagag ctttatgtgt gttatcatat tt#atgttttt  27840ttttcttttt tttttttttt gagaccgagt ttcactcttg ttgcccaagc tg#gagtgcaa  27900tggcacgatc tcggctcact gcaacctctg cccccaggtt caagtgattc tc#ctgcctca  27960 gcctcctgag tagctgggat tacaggcaca caccaccatg c    #                   #28001 <210> SEQ ID NO 4 <211> LENGTH: 520<212> TYPE: PRT <213> ORGANISM: Human <400> SEQUENCE: 4Met Pro Gly Ser Leu Pro Leu Asn Ala Glu Al #a Cys Trp Pro Lys Asp 1               5   #                10   #                15Val Gly Ile Val Ala Leu Glu Ile Tyr Phe Pr #o Ser Gln Tyr Val Asp            20       #            25       #            30Gln Ala Glu Leu Glu Lys Tyr Asp Gly Val As #p Ala Gly Lys Tyr Thr        35           #        40           #        45Ile Gly Leu Gly Gln Ala Lys Met Gly Phe Cy #s Thr Asp Arg Glu Asp    50               #    55               #    60Ile Asn Ser Leu Cys Met Thr Val Val Gln As #n Leu Met Glu Arg Asn65                   #70                   #75                   #80Asn Leu Ser Tyr Asp Cys Ile Gly Arg Leu Gl #u Val Gly Thr Glu Thr                85   #                90   #                95Ile Ile Asp Lys Ser Lys Ser Val Lys Thr As #n Leu Met Gln Leu Phe            100       #           105       #           110Glu Glu Ser Gly Asn Thr Asp Ile Glu Gly Il #e Asp Thr Thr Asn Ala        115           #       120           #       125Cys Tyr Gly Gly Thr Ala Ala Val Phe Asn Al #a Val Asn Trp Ile Glu    130               #   135               #   140Ser Ser Ser Trp Asp Gly Arg Tyr Ala Leu Va #l Val Ala Gly Asp Ile145                 1 #50                 1 #55                 1 #60Ala Val Tyr Ala Thr Gly Asn Ala Arg Pro Th #r Gly Gly Val Gly Ala                165   #               170   #               175Val Ala Leu Leu Ile Gly Pro Asn Ala Pro Le #u Ile Phe Glu Arg Gly            180       #           185       #           190Leu Arg Gly Thr His Met Gln His Ala Tyr As #p Phe Tyr Lys Pro Asp        195           #       200           #       205Met Leu Ser Glu Tyr Pro Ile Val Asp Gly Ly #s Leu Ser Ile Gln Cys    210               #   215               #   220Tyr Leu Ser Ala Leu Asp Arg Cys Tyr Ser Va #l Tyr Cys Lys Lys Ile225                 2 #30                 2 #35                 2 #40His Ala Gln Trp Gln Lys Glu Gly Asn Asp Ly #s Asp Phe Thr Leu Asn                245   #               250   #               255Asp Phe Gly Phe Met Ile Phe His Ser Pro Ty #r Cys Lys Leu Val Gln            260       #           265       #           270Lys Ser Leu Ala Arg Met Leu Leu Asn Asp Ph #e Leu Asn Asp Gln Asn        275           #       280           #       285Arg Asp Lys Asn Ser Ile Tyr Ser Gly Leu Gl #u Ala Phe Gly Asp Val    290               #   295               #   300Lys Leu Glu Asp Thr Tyr Phe Asp Arg Asp Va #l Glu Lys Ala Phe Met305                 3 #10                 3 #15                 3 #20Lys Ala Ser Ser Glu Leu Phe Ser Gln Lys Th #r Lys Ala Ser Leu Leu                325   #               330   #               335Val Ser Asn Gln Asn Gly Asn Met Tyr Thr Se #r Ser Val Tyr Gly Ser            340       #           345       #           350Leu Ala Ser Val Leu Ala Gln Tyr Ser Pro Gl #n Gln Leu Ala Gly Lys        355           #       360           #       365Arg Ile Gly Val Phe Ser Tyr Gly Ser Gly Le #u Ala Ala Thr Leu Tyr    370               #   375               #   380Ser Leu Lys Val Thr Gln Asp Ala Thr Pro Gl #y Ser Ala Leu Asp Lys385                 3 #90                 3 #95                 4 #00Ile Thr Ala Ser Leu Cys Asp Leu Lys Ser Ar #g Leu Asp Ser Arg Thr                405   #               410   #               415Gly Val Ala Pro Asp Val Phe Ala Glu Asn Me #t Lys Leu Arg Glu Asp            420       #           425       #           430Thr His His Leu Val Asn Tyr Ile Pro Gln Gl #y Ser Ile Asp Ser Leu        435           #       440           #       445Phe Glu Gly Thr Trp Tyr Leu Val Arg Val As #p Glu Lys His Arg Arg    450               #   455               #   460Thr Tyr Ala Arg Arg Pro Thr Pro Asn Asp As #p Thr Leu Asp Glu Gly465                 4 #70                 4 #75                 4 #80Val Gly Leu Val His Ser Asn Ile Ala Thr Gl #u His Ile Pro Ser Pro                485   #               490   #               495Ala Lys Lys Val Pro Arg Leu Pro Ala Thr Al #a Ala Glu Pro Glu Ala            500       #           505       #           510Ala Val Ile Ser Asn Gly Glu His         515           #       520<210> SEQ ID NO 5 <211> LENGTH: 518 <212> TYPE: PRT<213> ORGANISM: Human <400> SEQUENCE: 5Met Pro Gly Ser Leu Pro Leu Asn Ala Glu Al #a Cys Trp Pro Lys Asp 1               5   #                10   #                15Val Gly Ile Val Ala Leu Glu Ile Tyr Phe Pr #o Ser Gln Tyr Val Asp            20       #            25       #            30Gln Ala Glu Leu Glu Lys Tyr Asp Gly Val As #p Ala Gly Lys Tyr Thr        35           #        40           #        45Ile Gly Leu Gly Gln Ala Lys Met Gly Phe Cy #s Thr Asp Arg Glu Asp    50               #    55               #    60Ile Asn Ser Leu Cys Met Thr Val Val Gln As #n Leu Met Glu Arg Asn65                   #70                   #75                   #80Asn Leu Ser Tyr Asp Cys Ile Gly Arg Leu Gl #u Val Gly Thr Glu Thr                85   #                90   #                95Ile Ile Asp Lys Ser Lys Ser Val Lys Thr As #n Leu Met Gln Leu Phe            100       #           105       #           110Glu Glu Ser Gly Asn Thr Asp Ile Glu Gly Il #e Asp Thr Thr Asn Ala        115           #       120           #       125Cys Tyr Gly Gly Thr Ala Ala Val Phe Asn Al #a Val Asn Trp Ile Glu    130               #   135               #   140Ser Ser Ser Trp Asp Gly Arg Tyr Ala Leu Va #l Val Ala Gly Asp Ile145                 1 #50                 1 #55                 1 #60Ala Val Tyr Ala Thr Gly Asn Ala Arg Pro Th #r Gly Gly Val Gly Ala                165   #               170   #               175Val Ala Leu Leu Ile Gly Pro Asn Ala Pro Le #u Ile Phe Glu Arg Gly            180       #           185       #           190Leu Arg Gly Thr His Met Gln His Ala Tyr As #p Phe Tyr Lys Pro Asp        195           #       200           #       205Met Leu Ser Glu Tyr Pro Ile Val Asp Gly Ly #s Leu Ser Ile Gln Cys    210               #   215               #   220Tyr Leu Ser Ala Leu Asp Arg Cys Tyr Ser Va #l Tyr Cys Lys Lys Ile225                 2 #30                 2 #35                 2 #40His Ala Gln Trp Gln Lys Glu Ala Asn Asp As #n Asp Phe Thr Leu Asn                245   #               250   #               255Asp Phe Gly Phe Met Ile Phe His Ser Pro Ty #r Cys Lys Leu Val Gln            260       #           265       #           270Lys Ser Leu Ala Arg Met Leu Leu Asn Asp Ph #e Leu Asn Asp Gln Asn        275           #       280           #       285Arg Asp Lys Asn Ser Ile Tyr Ser Gly Leu Ly #s Ala Phe Gly Asp Val    290               #   295               #   300Lys Leu Glu Asp Thr Tyr Phe Asp Arg Asp Va #l Glu Lys Ala Phe Met305                 3 #10                 3 #15                 3 #20Lys Ala Ser Ser Glu Leu Phe Ser Gln Lys Th #r Lys Ala Ser Leu Leu                325   #               330   #               335Val Ser Asn Gln Asn Gly Asn Met Tyr Thr Se #r Ser Val Tyr Gly Ser            340       #           345       #           350Leu Ala Ser Val Leu Ala Gln Tyr Ser Pro Gl #n His Leu Ala Gly Lys        355           #       360           #       365Arg Ile Gly Val Phe Ser Tyr Gly Ser Gly Le #u Ala Ala Thr Leu Tyr    370               #   375               #   380Ser Leu Lys Val Thr Gln Asp Ala Thr Pro Gl #y Ser Ala Leu Asp Lys385                 3 #90                 3 #95                 4 #00Ile Thr Ala Ser Leu Cys Asp Leu Lys Ser Ar #g Leu Asp Ser Arg Thr                405   #               410   #               415Gly Val Ala Gln Asp Val Phe Ala Glu Asn Me #t Lys Leu Arg Glu Asp            420       #           425       #           430Thr His His Leu Val Asn Tyr Ile Pro Gln Gl #y Ser Ile Asp Ser Leu        435           #       440           #       445Phe Glu Gly Thr Trp Tyr Leu Val Arg Val As #p Glu Lys His Arg Arg    450               #   455               #   460Thr Tyr Ala Arg Arg Pro Thr Pro Asn Asp As #p Thr Leu Asp Glu Gly465                 4 #70                 4 #75                 4 #80Val Gly Leu Val His Ser Asn Ile Ala Thr Gl #u His Ile Pro Ser Pro                485   #               490   #               495Ala Lys Lys Val Pro Arg Leu Pro Ala Thr Al #a Ala Glu Pro Glu Ala            500       #           505       #           510Ala Val Ile Ser Asn Gly         515

That which is claimed is:
 1. An isolated polypeptide having an aminoacid sequence consisting of SEQ ID NO:2.
 2. An isolatedhydroxymethylglutaryl-CoA synthase having an amino acid sequencecomprising SEQ ID NO:2.
 3. A composition comprising the polypeptide ofclaim 1 and a carrier.
 4. A composition comprising thehydroxymethylglutaryl-CoA synthase of claim 2 and a carrier.